Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents.
Scope of review
The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed.
The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1 nm, but rarely better than 1 nm) method which has been used to discover the mechanics of a small protein, 53 kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14 Å-resolution IgG antibody three-dimensional map.
It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures.
protein structure; lipoprotein structure; electron microscopy; negative-staining; optimized negative-staining protocol; individual-particle electron tomography
Peptoids are a rapidly developing class of biomimetic polymers based on oligo-N-substituted glycine backbones, designed to mimic peptides and proteins. Inspired by natural antimicrobial peptides, a group of cationic amphipathic peptoids has been successfully discovered with a potent and broad-spectrum activity against pathogenic bacteria; however, there are limited studies to address the in vivo pharmacokinetics of the peptoids. Herein, 64Cu labeled DOTA conjugates of three different peptoids and two control peptides were synthesized and assayed in vivo by both biodistribution studies and small animal positron emission tomography (PET). The study was designed in a way to assess how structural differences of the peptidomimetics affect in vivo pharmacokinetics. As amphipathic molecules, major uptake of the peptoids occurred at the liver. Increased kidney uptake was observed by deleting one hydrophobic residue in the peptoid, and 64Cu-3 achieved the highest kidney uptake of all the conjugates tested in this study. In comparison to peptides, our data indicated that peptoids had general in vivo properties of higher tissue accumulation, slower elimination, and higher in vivo stability. Different administration routes (intravenous, intraperitoneal, and oral) were investigated with peptoids. When administered orally, the peptoids showed poor bioavailability, reminiscent to that of peptide. But, remarkably longer passage through the gastrointestinal (GI) tract without rapid digestion was observed for peptoids. These unique in vivo properties of peptoids were rationalized by efficient cellular membrane permeability and protease resistance of peptoids. The results observed in the biodistribution studies could be confirmed by the PET imaging, which provides a reliable way to evaluate in vivo pharmacokinetic properties of peptoids noninvasively and in real time. The pharmacokinetic data presented here can provide an insight for further development of the antimicrobial peptoids as pharmaceuticals.
Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.
AIM: To reveal the clinicopathological features and risk factors for lymph node metastases in gastric cardiac adenocarcinoma of male patients.
METHODS: We retrospective reviewed a total of 146 male and female patients with gastric cardiac adenocarcinoma who had undergone curative gastrectomy with lymphadenectomy in the Department of Surgery, Xin Hua Hospital and Rui Jin Hospital of Shanghai Jiaotong University Medical School between November 2001 and May 2012. Both the surgical procedure and extent of lymph node dissection were based on the recommendations of Japanese gastric cancer treatment guidelines. Univariate and multivariate analyses of lymph node metastases and the clinicopathological features were undertaken.
RESULTS: The rate of lymph node metastases in male patients with gastric cardiac adenocarcinoma was 72.1%. Univariate analysis showed an obvious correlation between lymph node metastases and tumor size, gross appearance, differentiation, pathological tumor depth, and lymphatic invasion in male patients. Multivariate logistic regression analysis revealed that tumor differentiation and pathological tumor depth were the independent risk factors for lymph node metastases in male patients. There was an obvious relationship between lymph node metastases and tumor size, gross appearance, differentiation, pathological tumor depth, lymphatic invasion at pN1 and pN2, and nerve invasion at pN3 in male patients. There were no significant differences in clinicopathological features or lymph node metastases between female and male patients.
CONCLUSION: Tumor differentiation and tumor depth were risk factors for lymph node metastases in male patients with gastric cardiac adenocarcinoma and should be considered when choosing surgery.
Gastric neoplasm; Lymph node metastasis; Risk factors; Gastrectomy; Lymphadenectomy
Brain glioma is a malignant tumor with a high incidence rate and poor prognosis that has become a focus of studies of central nervous system diseases. Previous studies have suggested that δ-opioid receptors may affect the proliferation and apoptosis of numerous types of tumor cells. However, to date, their precise mechanism(s) of action have not been elucidated. The present study aimed to investigate the effects of inhibiting δ-opioid receptors in brain glioma cell proliferation and apoptosis and their relevant molecular mechanisms. Various doses of naltrindole were supplied to treat brain glioma cells using the MTT method to assess the proliferation index. Flow cytometry was used to investigate the changes in cell apoptosis and mitochondrial membrane potential. The expression levels of Bax, Bcl-2, Bcl-xL, cytochrome c, caspase-9, caspase-3 and protein kinase C (PKC) were measured using western blotting. Naltrindole was observed to inhibit brain glioma cell proliferation and promote apoptosis in a dose- and time-dependent manner. Furthermore, the addition of naltrindole lead to changes in the brain glioma cell membrane potential and regulated Bax translocation to the mitochondrial membrane, consequently promoting the release of cytochrome c into the cytoplasm, followed by the activation of caspase-9 and -3, which caused cell apoptosis. In addition, naltrindole was able to regulate the expression levels of the cellular internal phosphorylated PKC proteins, which are closely associated with the inhibition of cell proliferation. In conclusion, the inhibition of δ-opioid receptors may inhibit brain glioma cell proliferation and lead to apoptosis, which is closely associated with the mitochondrial and PKC pathways.
δ-opioid receptor; glioma; apoptosis; mitochondria; protein kinase C pathway
The present work demonstrates that Cy5.5 conjugated Fe3O4/SiO2 core/shell nanoparticles could allow us to control movement of human natural killer cells (NK-92MI) by an external magnetic field. Required concentration of the nanoparticles for the cell manipulation is as low as ~20 μg Fe/mL. However, the relative ratio of the nanoparticles loaded NK-92MI cells infiltrated into the target tumor site is enhanced by 17-fold by applying magnetic field and their killing activity is still maintained as same as the NK-92MI cells without the nanoparticles. This approach allows us to open alternative clinical treatment with reduced toxicity of the nanoparticles and enhanced infiltration of immunology to the target site.
Fe3O4/SiO2 core/shell nanoparticles; Multifunctional nanoparticles; Magnetic field guided cell control; Natural killer cells; Tumor killing activity
Differentiation of Embryonic Stem Cells 1 (Dies1) was recently identified as a novel type I immunoglobulin (IgG) domain-containing plasma membrane protein important for effective differentiation of a murine pluripotent embryonic stem cell line. In this setting, Dies1 enhances bone morphogenetic protein 4 (BMP4) signaling. Here we show Dies1 transcript expression is induced ∼225-fold during in vitro adipogenesis of 3T3-L1 murine preadipocytes. Immunocytochemical imaging using ectopic expression of Flag-tagged Dies1 in 3T3-L1 adipocytes revealed localization to the adipocyte plasma membrane. Modulation of adipocyte phenotype with with tumor necrosis factor-α (TNFα) treatment or by siRNA knockdown of the master pro-adipogenic transcription factor peroxisome proliferator activated receptor gamma (PPARγ) resulted in a 90% and 60% reduction of Dies1 transcript levels, respectively. Moreover, siRNA-mediated Dies1 knockdown in 3T3-L1 preadipocytes inhibited adipogenic conversion. Such cultures had a 35% decrease in lipid content and a 45%–65% reduction in expression of key adipocyte transcripts, including that for PPARγ. The standard protocol for full in vitro adipogenic conversion of committed preadipocytes, such as 3T3-L1, does not include BMP4 treatment. Thus we posit the positive role of Dies1 in adipogenesis, unlike that for Dies1 in differentiation of embryonic stem cells, does not include its pro-BMP4 effects. In support of this idea, 3T3-L1 adipocytes knocked down for Dies1 did not evidence decreased phospho-Smad1 levels upon BMP4 exposure. qPCR analysis of Dies1 transcript in multiple murine and human tissues reveals high enrichment in white adipose tissue (WAT). Interestingly, we observed a 10-fold induction of Dies1 transcript in WAT of fasted vs. fed mice, suggesting a role for Dies1 in nutritional response of mature fat cells in vivo. Together our data identify Dies1 as a new differentiation-dependent adipocyte plasma membrane protein whose expression is required for effective adipogenesis and that may also play a role in regard to nutritional status in WAT.
AIM: To explore risk factors for lymph node metastases in early gastric cancer (EGC) and to confirm the appropriate range of lymph node dissection.
METHODS: A total of 202 patients with EGC who underwent curative gastrectomy with lymphadenectomy in the Department of Surgery, Xinhua Hospital and Ruijin Hospital of Shanghai Jiaotong University Medical School between November 2003 and July 2009, were retrospectively reviewed. Both the surgical procedure and the extent of lymph node dissection were based on the recommendations of the Japanese gastric cancer treatment guidelines. The macroscopic type was classified as elevated (type I or IIa), flat (IIb), or depressed (IIc or III). Histopathologically, papillary and tubular adenocarcinomas were grouped together as differentiated adenocarcinomas, and poorly differentiated and signet-ring cell adenocarcinomas were regarded as undifferentiated adenocarcinomas. Univariate and multivariate analyses of lymph node metastases and patient and tumor characteristics were undertaken.
RESULTS: The lymph node metastases rate in patients with EGC was 14.4%. Among these, the rate for mucosal cancer was 5.4%, and 8.9% for submucosal cancer. Univariate analysis showed an obvious correlation between lymph node metastases and tumor location, depth of invasion, morphological classification and venous invasion (χ2 = 122.901, P = 0.001; χ2 = 7.14, P = 0.008; χ2 = 79.523, P = 0.001; χ2 = 8.687, P = 0.003, respectively). In patients with submucosal cancers, the lymph node metastases rate in patients with venous invasion (60%, 3/5) was higher than in those without invasion (20%, 15/75) (χ2 = 4.301, P = 0.038). Multivariate logistic regression analysis revealed that the depth of invasion was the only independent risk factor for lymph node metastases in EGC [P = 0.018, Exp (B) = 2.744]. Among the patients with lymph node metastases, 29 cases (14.4%) were at N1, seven cases were at N2 (3.5%), and two cases were at N3 (1.0%). Univariate analysis of variance revealed a close relationship between the depth of invasion and lymph node metastases at pN1 (P = 0.008).
CONCLUSION: The depth of invasion was the only independent risk factor for lymph node metastases. Risk factors for metastases should be considered when choosing surgery for EGC.
Gastric neoplasm; Lymph node metastasis; Risk factors; Gastrectomy; Lymphadenectomy
AIM: To review the clinicopathological characteristics of concurrent gastrointestinal stromal tumors (GISTs) and gastric adenocarcinoma.
METHODS: We retrospectively analyzed eight cases of synchronous adenocarcinoma and GIST in the stomach that had been surgically resected with curative intent between March 2003 and December 2008 in Xinhua hospital and Ruijin hospital. The adenocarcinoma was determined to be the primary tumor based on the histological features. The GIST cells were diffusely and strongly positive for CD34 and CD117.
RESULTS: The patients were six men and two women aged 47-80 years (average, 68.6 years). GIST was preoperatively detected in only one patient. The average sizes of the gastric adenocarcinomas and GISTs were 6.000 ± 2.6186 cm and 1.825 ± 1.4370 cm, respectively. All GISTs were very low- or low-risk lesions that were detected during evaluation, staging, operation or follow-up for gastric adenocarcinoma.
CONCLUSION: We hypothesized that the stomach was influenced by the same unknown carcinogen, resulting in a simultaneous proliferation of different cell lines (epithelial and stromal cell).
Gastric adenocarcinoma; Gastrointestinal stromal tumor; Synchronous occurrence; Gastrectomy
Activation of interferon (IFN) signaling in the central nervous system (CNS) is usually associated with inflammation. However, a robust activation of type I IFN-stimulated genes (ISGs) at pre-symptomatic stages occurs in the spinal cord of SOD1(G93A) mice, an amyotrophic lateral sclerosis (ALS) animal model, without obvious signs of inflammation. To determine if the same signaling pathway is elevated in other types of neuronal injuries, we examined the protein expression levels of an IFN-stimulated gene, ISG15, in mouse models of acute and chronic neuronal injuries. We found that ISG15 protein was dramatically increased in the brains of mice subjected to global ischemia and traumatic brain injury, and in transgenic mice overexpressing HIV gp120 protein. These results suggest that activation of ISGs is a shared feature of neuronal injuries and that ISG15 may be a suitable biomarker for detecting neuronal injuries in the CNS.
interferon-stimulated gene 15; neuronal injury; biomarker
Peptides show much promise as potent and selective drug candidates. Fusing peptides to a scaffold monoclonal antibody produces a conjugated antibody which has the advantages of peptide activity yet also has the pharmacokinetics determined by the scaffold antibody. However, the conjugated antibody often has poor binding affinity to antigens that may be related to unknown structural changes. The study of the conformational change is difficult by conventional techniques because structural fluctuation under equilibrium results in multiple structures co-existing. Here, we employed our two recently developed electron microscopy (EM) techniques: optimized negative-staining (OpNS) EM and individual-particle electron tomography (IPET). Two-dimensional (2D) image analyses and three-dimensional (3D) maps have shown that the domains of antibodies present an elongated peptide-conjugated conformational change, suggesting that our EM techniques may be novel tools to monitor the structural conformation changes in heterogeneous and dynamic macromolecules, such as drug delivery vehicles after pharmacological synthesis and development.
Preoperative characterization of complex solid and cystic adnexal masses is crucial for informing patients about possible surgical strategies. Our study aims to determine the usefulness of apparent diffusion coefficients (ADC) for characterizing complex solid and cystic adnexal masses.
One-hundred and 91 patients underwent diffusion-weighted (DW) magnetic resonance (MR) imaging of 202 ovarian masses. The mean ADC value of the solid components was measured and assessed for each ovarian mass. Differences in ADC between ovarian masses were tested using the Student’s t-test. The receiver operating characteristic (ROC) was used to assess the ability of ADC to differentiate between benign and malignant complex adnexal masses.
Eighty-five patients were premenopausal, and 106 were postmenopausal. Seventy-four of the 202 ovarian masses were benign and 128 were malignant. There was a significant difference between the mean ADC values of benign and malignant ovarian masses (p < 0.05). However, there were no significant differences in ADC values between fibrothecomas, Brenner tumors and malignant ovarian masses. The ROC analysis indicated that a cutoff ADC value of 1.20 x10-3 mm2/s may be the optimal one for differentiating between benign and malignant tumors.
A high signal intensity within the solid component on T2WI was less frequently in benign than in malignant adnexal masses. The combination of DW imaging with ADC value measurements and T2-weighted signal characteristics of solid components is useful for differentiating between benign and malignant ovarian masses.
Ovary; Ovarian tumors; Diffusion-weighted imaging; Apparent diffusion coefficients
Gastrointestinal stromal tumors (GISTs) are nonepithelial, mesenchymal neoplasms that rarely occur in children.
We present a unique case of a GIST that developed outside the gastrointestinal tract within the mesoappendix of a 6-year old boy. Computed tomography (CT) revealed a slightly lobulated, homogeneous soft-tissue mass, with marked contrast enhancement.
This case study provides new insight into the CT appearance of extragastrointestinal stromal tumors.
Children; CT; Extragastrointestinal stromal tumor
Recent high-throughput-sequencing of the cancer genome has identified oncogenic mutations in BRaf genetic locus as one of the critical events in melanomagenesis. In normal cells, the activity of BRaf is tightly regulated. Gain-of-function mutations like those identified in melanoma frequently lead to enhanced cell-survival and unrestrained growth. The activating mutation of BRaf will also induce the cells to senesce. However, the mechanism by which the oncogenic BRaf induces the senescent barrier remains poorly defined. microRNAs have regulatory functions toward the expression of genes that are important in carcinogenesis. Here we show that expression of several microRNAs is altered when the oncogenic version of BRaf is introduced in cultured primary melanocytes and these cells undergo premature cellular senescence. These include eight microRNAs whose expression rates are significantly stimulated and three that are repressed. While most of the induced microRNAs have documented negative effects on cell cycle progression, one of the repressed microRNAs has proven oncogenic functions. Ectopic expression of some of these induced microRNAs increased the expression of senescence markers and induced growth arrest and senescence in primary melanocytes. Taken together, our results suggest that the change in microRNA expression rates may play a vital role in senescence induced by the oncogenic BRaf.
Agouti-related protein (AgRP) is a 4-kDa cystine-knot peptide of human origin with four disulfide bonds and four solvent-exposed loops. The cell adhesion receptor integrin αvβ3 is an important tumor angiogenesis factor that determines the invasiveness and metastatic ability of many malignant tumors. AgRP mutants have been engineered to bind to integrin αvβ3 with high affinity and specificity using directed evolution. Here, AgRP mutants 7C and 6E were radiolabeled with 111In and evaluated for in vivo targeting of tumor integrin αvβ3 receptors. AgRP peptides were conjugated to the metal chelator 1, 4, 7, 10-tetra-azacyclododecane- N, N′, N″, N‴-tetraacetic acid (DOTA) and radiolabeled with 111In. The stability of the radiopeptides 111In-DOTA-AgRP-7C and 111In-DOTA-AgRP-6E was tested in phosphate-buffered saline (PBS) and mouse serum, respectively. Cell uptake assays of the radiolabeled peptides were performed in U87MG cell lines. Biodistribution studies were performed to evaluate the in vivo performance of the two resulting probes using mice bearing integrin-expressing U87MG xenograft tumors. Both AgRP peptides were easily labeled with 111In in high yield and radiochemical purity (>99%). The two probes exhibited high stability in phosphate-buffered saline and mouse serum. Compared with 111In-DOTA-AgRP-6E, 111In-DOTA-AgRP-7C showed increased U87MG tumor uptake and longer tumor retention (5.74 ± 1.60 and 1.29 ± 0.02%ID/g at 0.5 and 24 h, resp.), which was consistent with measurements of cell uptake. Moreover, the tumor uptake of 111In-DOTA-AgRP-7C was specifically inhibited by coinjection with an excess of the integrin-binding peptidomimetic c(RGDyK). Thus, 111In-DOTA-AgRP-7C is a promising probe for targeting integrin αvβ3 positive tumors in living subjects.
Affibody molecules have received significant attention in the fields of molecular imaging and drug development. However, Affibody scaffolds display an extremely high renal uptake, especially when modified with chelators and then labeled with radiometals. This unfavorable property may impact their use as radiotherapeutic agents in general and as imaging probes for the detection of tumors adjacent to kidneys in particular. Herein, we present a simple and generalizable strategy for reducing the renal uptake of Affibody molecules while maintaining their tumor uptake. Human serum albumin (HSA) was consecutively modified by 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS ester) and the bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC). The HER2 Affibody analog, Ac-Cys-ZHER2:342, was covalently conjugated with HSA, and the resulting bioconjugate DOTA-HSA-ZHER2:342 was further radiolabeled with 64Cu and 111In and evaluated in vitro and in vivo. Radiolabeled DOTA-HSA-ZHER2:342 conjugates displayed a significant and specific cell uptake into SKOV3 cell cultures. Positron emission tomography (PET) investigations using 64Cu-DOTA-HSA-ZHER2:342 were performed in SKOV3 tumor-bearing nude mice. High tumor uptake values (> 14% ID/g at 24 h and 48 h) and high liver accumulations but low kidney accumulations were observed. Biodistribution studies and single-photon emission computed tomography (SPECT) investigations using 111In-DOTA-HSA-ZHER2:342 validated these results. At 24 h post injection, the bio distribution data revealed high tumor (16.26% ID/g) and liver uptake (14.11% ID/g) but relatively low kidney uptake (6.06% ID/g). Blocking studies with co-injected, non-labeled Ac-Cys-ZHER2:342 confirmed the in vivo specificity of HER2. Radiolabeled DOTA-HSA-ZHER2:342 Affibody conjugates are promising SPECT and PET-type probes for the imaging of HER2 positive cancer. More importantly, DOTA-HSA-ZHER2:342 is suitable for labeling with therapeutic radionuclides (e.g. 90Y or 177Lu) for treatment studies. The approach of using HSA to optimize the pharmacokinetics and biodistribution profile of Affibodies, may be extended to the design of many other targeting molecules.
This article reports the affibody-based nanoprobes specifically target and image human epidermal growth factor receptor type 2 (HER2)-expressing cells and tumors. The simple, robust, and precise structure of affibody molecules are a promising class of targeting ligands with high affinity. Using near-infrared (NIR) quantum dots (QDs) and iron oxide (IO) nanoparticles as two representative nanomaterials, we designed anti–HER2 affibody molecules with an N-terminus cysteine residue (Cysteine-ZHER2:342) and precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates. The in vitro and in vivo study showed the conjugates are highly specific to target and image HER2-expressing cells and tumors. This work indicated the nanoparticle-affibody conjugates may be excellent candidates as targeting probes for molecular imaging and diagnosis.
Affibody; bioconjugation; nanoprobes; HER2; molecular imaging
The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.
Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP’s function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.
Raf kinase inhibitor protein; Raf/MEK/ERK, NF-κB, matrix metalloproteinases; cancer cell migration, invasion and metastasis
In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several alpha-melanocyte-stimulating hormone (α-MSH) analogs have been labeled with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-18F-fluoropropionate (18F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma.
Metallopeptides Ac-d,Lys-ReCCMSH(Arg11) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with 19F-NFP or 18F-NFP. The resulting cold or radiofluorinated metallopeptides, 18/19F-FP-RMSH-1 and 18/19F-FP-RMSH-2 were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution and small-animal PET imaging properties.
The binding affinities of the 19F-FP-RMSH-1 and 19F-FP-RMSH-2) were determined to be within low nM range. In vivo studies revealed that both 18F-labeled metallopeptides possessed good tumor uptake in B16F10 murine model with high MC1R expression, while much lower uptake in A375M human melanoma xenografts. Moreover, 18F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in kidney and liver, when compared to 18F-FP-RMSH-2 at 2 h post-injection (p.i.). 18F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with the same peptide labeled with 18F-SFB (named as 18F-FB-RMSH-1). Small animal PET imaging of 18F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organs contrast were observed for A375M model than that of B16F10 model. 18F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with 18F-FB-RMSH-1.
18F-FP-RMSH-1 demonstrates significant advantages over 18F-FB-RMSH-1 and 18F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo.
α-MSH; MC1R; PET; Malignant Melanoma; Metallopeptide, 18F
The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with 64Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.
DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.
Methylation-sensitive amplified polymorphism; Epigenetic modification; Tobacco mosaic virus (TMV) resistance; Flue-cured tobacco
Microglial activation plays an important role in neurodegenerative diseases through production of nitric oxide (NO) and several pro-inflammatory cytokines. Lipoxins (LXs) and aspirin-triggered LXs (ATLs) are considered to act as 'braking signals' in inflammation. In the present study, we investigated the effect of aspirin-triggered LXA4 (ATL) on infiammatory responses induced by lipopolysaccharide (LPS) in murine microglial BV-2 cells.
BV-2 cells were treated with ATL prior to LPS exposure, and the effects of such treatment production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) were analysed by Griess reaction, ELISA, western blotting and quantitative RT-PCR. Moreover, we investigated the effects of ATL on LPS-induced nuclear factor-κB (NF-κB) activation, phosphorylation of mitogen-activated protein kinases (MAPKs) and activator protein-1 (AP-1) activation.
ATL inhibited LPS-induced production of NO, IL-1β and TNF-α in a concentration-dependent manner. mRNA expressions for iNOS, IL-1β and TNF-α in response to LPS were also decreased by ATL. These effects were inhibited by Boc-2 (a LXA4 receptor antagonist). ATL significantly reduced nuclear translocation of NF-κB p65, degradation of the inhibitor IκB-α, and phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in BV-2 cells activated with LPS. Furthermore, the DNA binding activity of NF-κB and AP-1 was blocked by ATL.
This study indicates that ATL inhibits NO and pro-inflammatory cytokine production at least in part via NF-κB, ERK, p38 MAPK and AP-1 signaling pathways in LPS-activated microglia. Therefore, ATL may have therapeutic potential for various neurodegenerative diseases.
Berberine (BBR) is a drug with multiple effects on cellular energy metabolism. The present study explored answers to the question of which CYP450 (Cytochrome P450) isoenzymes execute the phase-I transformation for BBR, and what are the bioactivities of its metabolites on energy pathways.
BBR metabolites were detected using LC-MS/MS. Computer-assistant docking technology as well as bioassays with recombinant CYP450s were employed to identify CYP450 isoenzymes responsible for BBR phase-I transformation. Bioactivities of BBR metabolites in liver cells were examined with real time RT-PCR and kinase phosphorylation assay.
In rat experiments, 4 major metabolites of BBR, berberrubine (M1), thalifendine (M2), demethyleneberberine (M3) and jatrorrhizine (M4) were identified in rat's livers using LC-MS/MS (liquid chromatography-tandem mass spectrometry). In the cell-free transformation reactions, M2 and M3 were detectable after incubating BBR with rCYP450s or human liver microsomes; however, M1 and M4 were below detective level. CYP2D6 and CYP1A2 played a major role in transforming BBR into M2; CYP2D6, CYP1A2 and CYP3A4 were for M3 production. The hepatocyte culture showed that BBR was active in enhancing the expression of insulin receptor (InsR) and low-density-lipoprotein receptor (LDLR) mRNA, as well as in activating AMP-activated protein kinase (AMPK). BBR's metabolites, M1-M4, remained to be active in up-regulating InsR expression with a potency reduced by 50-70%; LDLR mRNA was increased only by M1 or M2 (but not M3 and M4) with an activity level 35% or 26% of that of BBR, respectively. Similarly, AMPK-α phosphorylation was enhanced by M1 and M2 only, with a degree less than that of BBR.
Four major BBR metabolites (M1-M4) were identified after phase-I transformation in rat liver. Cell-free reactions showed that CYP2D6, CYP1A2 and CYP3A4 seemed to be the dominant CYP450 isoenzymes transforming BBR into its metabolites M2 and M3. BBR's metabolites remained to be active on BBR's targets (InsR, LDLR, and AMPK) but with reduced potency.
Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.
lipoprotein structure; lipoprotein morphology; protocol