E0771 and 4T1 murine breast cancer cells and interleukin 6 alter gene expression patterns but do not induce browning in cultured white adipocytes

Breast cancer remains a substantial clinical problem worldwide, and cancer-associated cachexia is a condition associated with poor prognosis in this and other malignancies. Adipose tissue is involved in the development and progression of cancer-associated cachexia, but its various roles and mechanisms of action are not completely defined, especially as it relates to breast cancer. Interleukin 6 has been implicated in several mechanisms contributing to increased breast cancer tumorigenesis, as well as a net-negative energy balance and cancer-associated cachexia via adipose tissue remodeling in other models of cancer; however, its potential role in breast cancer-associated white adipose browning has not been explored. In this study, we demonstrate localized white adipose tissue browning in a spontaneous model of murine mammary cancer. We then used an in vitro murine adipocyte culture system with the E0771 and 4T1 cell lines as models of breast cancer. We demonstrate that while the E0771 and 4T1 secretomes and cross-talk with white adipocytes alter white adipocyte mRNA expression, they do not directly induce white adipocyte browning. Additionally, we show that neither exogenous administration of interleukin 6 alone or with its soluble receptor directly induce white adipocyte browning. Together, these results demonstrate that neither the E0771 or 4T1 murine breast cancer cell lines, nor interleukin 6, directly cause browning of cultured white adipocytes. This suggests that their roles in adipose tissue remodeling are more complex and indirect in nature.     

Intravascular heavy chain-modification of hyaluronan during endotoxic shock

During inflammation, the covalent linking of the ubiquitous extracellular polysaccharide hyaluronan (HA) with the heavy chains (HC) of the serum protein inter alpha inhibitor (IαI) is exclusively mediated by the enzyme tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6). While significant advances have been made regarding how HC-modified HA (HC-HA) is an important regulator of inflammation, it remains unclear why HC-HA plays a critical role in promoting survival in intraperitoneal lipopolysaccharide (LPS)-induced endotoxemia while exerting only a modest role in the outcomes following intratracheal exposure to LPS. To address this gap, the two models of intraperitoneal LPS-induced endotoxic shock and intratracheal LPS-induced acute lung injury were directly compared in TSG-6 knockout mice and littermate controls. HC-HA formation, endogenous TSG-6 activity, and inflammatory markers were assessed in plasma and lung tissue. TSG-6 knockout mice exhibited accelerated mortality during endotoxic shock. While both intraperitoneal and intratracheal LPS induced HC-HA formation in lung parenchyma, only systemically-induced endotoxemia increased plasma TSG-6 levels and intravascular HC-HA formation. Cultured human lung microvascular endothelial cells secreted TSG-6 in response to both TNFα and IL1β stimulation, indicating that, in addition to inflammatory cells, the endothelium may secrete TSG-6 into circulation during systemic inflammation. These data show for the first time that LPS-induced systemic inflammation is uniquely characterized by significant vascular induction of TSG-6 and HC-HA, which may contribute to improved outcomes of endotoxemia.     

lpp deletion as a permeabilization method

Our earlier studies with outer membrane permeability in E. coli showed that an insertion mutation in lpp gene (encoding Braun's lipoprotein) drastically changed the outer membrane permeability, resulting in significant acceleration of whole-cell catalyzed reactions. In order to gain a mechanistic understanding of the nature of permeability change, the lpp region was sequenced. The results revealed that Lpp was not expressed in the insertion mutant, suggesting that the absence, rather than the alteration, of Lpp is responsible for the observed permeability change. This surprising result prompts us to investigate the possibility of establishing lpp deletion as a general permeabilization method. Two lpp deletion mutants were generated from strains with different genetic background and the effect of lpp deletion on cell physiology was investigated. While lpp deletion had no significant effect on cell growth, carbon metabolism, and fatty acid compositions, it enhanced permeability of various small molecules, consistent with the results with the insertion mutant. This phenotype is useful in a wide range of biotechnological applications. We illustrate here the use of the mutant with organophosphate hydrolysis and L-carnitine synthesis, where permeability is known to be a limiting factor. Both processes were significantly improved with the mutant because of enhanced permeability through the outer membrane. Therefore, this study has established an easy yet generally applicable method for permeabilizing E. coli cells without significant adverse effects. Further, as lpp homolog is known to exist in gram-negative bacteria, we expect that this method will be applicable to other gram-negative bacteria.     

Polymorphism attribution of cSNPs in cancer-related genes located in loss regions with a high frequency of HCC between HBV and health groups

Cancer-related genes harbored in the loss regions containing a high frequency of hepatocellular carcinoma (HCC) were selected. Related information was gathered and the coding single nucleotide polymorphism (cSNP) sequences were obtained from the single nucleotide polymorphism (SNP) database. The appropriate primers and oligonucleotide probes were then designed in accordance with the SNP sites, and subsequently, the gene chips for detecting SNPs were constructed. Genomic DNA was extracted from blood samples of healthy controls and from patients with HBV infection. The sequences, including the SNPs, were amplified via polymerase chain reaction (PCR) and labeled using digoxigenin deoxyuridine tri-phosphate (Dig-dUTP). The labeled products were then hybridized with the SNP chips. Results confirmed that the differences in allele frequencies of three SNPs EGFL3 (rs947345), Caspase9 (rs2308950), and E2F2 (rs3218171) were distinct between HBV-infected patients and controls, suggesting that these SNPs ocuring in high frequency in HBV-infected individuals may be associated with susceptibility to HCC. Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2006, 39(3): 1–5 [译自: 南开大学学报(自然科学版)]     

Oleanolic Acid Induces Apoptosis in Human Leukemia Cells through Caspase Activation and Poly（ADP-ribose） Polymerase Cleavage

It has been shown that Fructus Ligustri Lucidi （FLL）, a promising traditional Chinese medicine, can inhibit the growth of tumors. However, the effective component and molecular mechanism of FLL act to inhibit tumor proliferation are unclear. In this study, we demonstrated that oleanolic acid （OA）, a principal chemical component of FLL, inhibited the proliferation of human leukemia HL60 cells in culture. MTT assay showed that treatment of HL60 cells with FLL crude extracts or OA dramatically blocked the growth of target tumor cell in a time- and dose-dependent manner. Morphological changes of the nuclei and DNA fragmentation showed that apoptotic cell death occurred in the HL60 cells after treating with FLL extracts （20 mg/ml） or OA （3.65×10^-2 mg/ml）. Furthermore, flow cytometry assay showed that treatment of HL60 cells with FLL or OA caused an increased accumulation of G1 and sub-G1 subpopulations. Western blot analysis showed that caspase-9 and caspase-3 were activated, accompanied by the cleavage of poly （ADP-ribose） polymerase （PARP） in the target cells during FLL- or OA-induced apoptosis, These results suggest that OA acts as the effective component of FLL by exerting its cytotoxicity towards target tumor cells through activation of caspases and cleavage of PARP.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Comparative Endocranial Anatomy,Encephalization, and Phylogeny of Notoungulata (Placentalia,Mammalia)

Journal of Mammalian Evolution - Cranial endocasts are one of the most direct tools available to obtain information about the endocranial cavity of fossil mammals, but few anatomical comparisons...