Determination of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea in plasma by negative chemical ionization mass spectrometry

A sensitive and specific method for the quantitative determination of a new antitumor agent, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea, (PCNU) has been developed for the analysis of plasma. This assay involves extraction of the plasma sample, separation of the drug by thin-layer chromatography and mass spectrometric detection of the negative ions derived from the unchanged drug and its 2H4-labeled analog. Ion current profile peaks are obtained when drug samples are introduced into the heated source using a desorption chemical ionization probe. Detection limits are below 1.0 ng ml-1 of plasma. This method has been applied to determine the in vitro rate of decomposition of PCNU in plasma and the plasma clearance of this drug from a cancer patient.     

Heterogeneity in the physiological states and pharmacological responses of differentiating 3T3-L1 preadipocytes

Increases in key components of adipogenesis and lipolysis pathways correlate at the population-averaged level during adipogenesis. However, differentiating preadipocytes are highly heterogeneous in cellular and lipid droplet (LD) morphologies, and the degree to which individual cells follow population-averaged trends is unclear. In this study, we analyze the molecular heterogeneity of differentiating 3T3-L1 preadipocytes using immunofluorescence microscopy. Unexpectedly, we only observe a small percentage of cells with high simultaneous expression of markers for adipogenesis (peroxisome proliferator-activated receptor γ [PPARγ], CCAAT/enhancer-binding protein α, and adiponectin) and lipid accumulation (hormone-sensitive lipase, perilipin A, and LDs). Instead, we identify subpopulations of cells with negatively correlated expressions of these readouts. Acute perturbation of adipocyte differentiation with PPARγ agonists, forskolin, and fatty acids induced subpopulation-specific effects, including redistribution of the percentage of cells in observed subpopulations and differential expression levels of PPARγ. Collectively, our results suggested that heterogeneity observed during 3T3-L1 adipogenesis reflects a dynamic mixture of subpopulations with distinct physiological states.     

Thermal-Stable Proteins of Fruit of Long-Living Sacred Lotus Nelumbo nucifera Gaertn var. China Antique

Single-seeded fruit of the sacred lotus Nelumbo nucifera Gaertn var. China Antique from NE China have been shown to remain viable for as long as ~1,300 years, determined by direct radiocarbon-dating, and to have a germination rate of 84 %. The pericarp, a fruit tissue that encloses the single seeds of Nelumbo, is one of the major factors contributing to fruit longevity. Proteins that are heat stable and have a protective function are equally important to such centuries-long seed viability. We document proteins of Nelumbo fruit that are able to withstand heating, 32 % of which remained soluble in the 110 °C-treated embryo axis of a 549-year-old fruit and 76 % retained fluidity in its cotyledons. The genome of Nelumbo has recently been published and annotated. The amino-acid sequences of 11 “thermal proteins” (soluble at 100 °C) of modern Nelumbo embryo axes and cotyledons, identified by mass spectrometry, Western blot and bioassay, are assembled and aligned with those of an archaeal hyperthermophile Methancaldococcus jannaschii (“Mj,” an anaerobic methanogen having a growth optimum of 85 °C) and with those of five mesophile angiosperms. These thermal proteins have roles in protection and repair under stress. More than half (55 %) of the durable Nelumbo thermal proteins are present in the archaean Mj, indicating their ancient history. One Nelumbo protein-repair enzyme exhibits activity at 100 °C, having a heat-tolerance higher than the comparable enzyme of Arabidopsis. A list of 30 sequenced but unassembled thermal proteins of Nelumbo is appended.     

Distinct Patterns of Stromal and Tumor Expression of ROR1 and ROR2 in Histological Subtypes of Epithelial Ovarian Cancer

OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients (n = 178). Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.