Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

Arabidopsis thaliana cdd1 mutant uncouples the constitutive activation of salicylic acid signalling from growth defects

Arabidopsis genotypes with a hyperactive salicylic acid-mediated signalling pathway exhibit enhanced disease resistance, which is often coupled with growth and developmental defects, such as dwarfing and spontaneous necrotic lesions on the leaves, resulting in reduced biomass yield. In this article, we report a novel recessive mutant of Arabidopsis, cdd1 (constitutive defence without defect in growth and development1), that exhibits enhanced disease resistance associated with constitutive salicylic acid signalling, but without any observable pleiotropic phenotype. Both NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1)-dependent and NPR1-independent salicylic acid-regulated defence pathways are hyperactivated in cdd1 mutant plants, conferring enhanced resistance against bacterial pathogens. However, a functional NPR1 allele is required for the cdd1-conferred heightened resistance against the oomycete pathogen Hyaloperonospora arabidopsidis. Salicylic acid accumulates at elevated levels in cdd1 and cdd1 npr1 mutant plants and is necessary for cdd1-mediated PR1 expression and disease resistance phenotypes. In addition, we provide data which indicate that the cdd1 mutation negatively regulates the npr1 mutation-induced hyperactivation of ethylene/jasmonic acid signalling.     

Expression of gibberellin mutations in fruits of Pisum sativum L.

The gibberellin (GA) economy of young pea (Pisum sativum L.) fruits was investigated using a range of mutants with altered GA biosynthesis or deactivation. The synthesis mutation lh-2 substantially reduced the content of both GA₄ and GA₁ in young seeds. Among the other synthesis mutations, ls-1, le-1 and le-3, the largest reduction in seed GA₁ content was only 1.7-fold (le-1), while GA₄ was not reduced in these mutants, and in fact accumulated in some experiments (compared with the wild type). Mutation sln appeared to block the step GA₂₀ to GA₂₉ in young pods and seeds, but not as strongly as in older seeds. Mutations ls-1, le-1 and le-3 markedly reduced pod GA₁ levels, but pod elongation was not affected. After feeds of [¹³C,³H]GA₂₀ to leaves, the pods contained ¹³C,³H-labelled GA₂₀, GA₁, GA₂₉ and GA₈₁, and the seeds, [¹³C,³H]GA₂₀ and [¹³C,³H]GA₂₉. These findings are discussed in relation to recent suggestions regarding the role and origin of GA₁ in pea fruits. Received: 6 June 1997 / Accepted: 15 July 1997     

The ghost of fouling communities past: the effect of original community on subsequent recruitment

Biofouling on ships has been linked to the spread of invasive species, which has been identified as one of the current primary threats to the environment. Previous research on antifouling coatings suggested that the quantity of fouling, as well as community composition, on biocidal coatings was modified by prior fouling settlement. The experiment reported in this paper was designed to determine how preconditioning affected the rate and composition of subsequent fouling on transplanted silicone coatings. A series of 10 × 20?cm panels coated with Intersleek 700 or DC3140 were placed at three locations in Florida (Ponce Inlet, Sebastian Inlet, and Port of Miami), which were characterized by distinct fouling communities. Panels were immersed for four months, cleaned, and reciprocally transplanted among the three sites. Fouling community composition and coverage were characterized at bimonthly intervals both before and after transplantation. The original fouling community affected the subsequent fouling composition and recolonization by tunicates, sea anemones, barnacles, sponges, hydroids, and arborescent bryozoans. The community-level effects were short-term, lasting 2–4?months, but specific responses lasted up to 14?months post-transplant.     

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the βA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.     

Lymphokine-mediated activation of a T cell-dependent IgA antipolysaccharide response

Immune responses to bacterial polysaccharides are important to host immunity at mucosal surfaces. We previously showed that BALB/c mice produce substantial T cell-dependent IgA responses to alpha (1,3) glucan determinants on the bacterial capsular polysaccharide dextran B1355. The data in this study demonstrate that the requirement for T cells for the activation of the IgA anti-alpha (1,3) dextran B1355 response can be replaced by T cell-derived nonantigen specific helper factors that appear to act during the late stages of B cell differentiation. Supernatants from the activated T cell lines cr-15 and (DL)C.C3.11.75, which contain interferon and late-acting T cell replacing factor activity, supported terminal differentiation of dextran-stimulated B cells to IgA anti-alpha (1,3) glucan antibody-forming cells and substantially increased IgM anti-alpha (1,3) glucan responses in culture. Although supernatants with interleukin 2 activity did not support optimal antigen-driven plaque-forming cell responses, they synergized with supernatants having interferon and T cell replacing factor activity in the production of IgA and IgM anti-alpha (1,3) glucan responses and IgM anti-SRBC responses. Supernatants from the T cell lines B6.11 and (DL)A.4 contained B cell growth factor activity but did not support activation of IgA anti-alpha (1,3) glucan PFC. These studies suggest that interferon and/or T cell replacing factor play an important role in the antigen-driven differentiation of B cells of the IgA and IgM isotypes to antibody-forming cells.     

Effects of Diet Composition on Postprandial Energy Availability during Weight Loss Maintenance

Background

The major circulating metabolic fuels regulate hunger, and each is affected by dietary composition. An integrated measure of postprandial energy availability from circulating metabolic fuels may help inform dietary recommendations for weight maintenance after weight loss.

Aim

We examined the effect of low-fat (LF, 60% of energy from carbohydrate, 20% fat, 20% protein), low-glycemic index (LGI, 40%–40%-20%), and very low-carbohydrate (VLC, 10%–60%-30%) diets on total postprandial metabolic fuel energy availability (EA) during weight loss maintenance.

Methods

Eight obese young adults were fed a standard hypocaloric diet to produce 10–15% weight loss. They were then provided isocaloric LF, LGI, and VLC diets in a randomized crossover design, each for a 4-week period of weight loss maintenance. At the end of each dietary period, a test meal representing the respective diet was provided, and blood samples were obtained every 30 minutes for 5 hours. The primary outcome was EA, defined as the combined energy density (circulating level×relative energy content) of glucose, free fatty acids, and β-hydroxybutyrate. Secondary outcomes were individual metabolic fuels, metabolic rate, insulin, glucagon, cortisol, epinephrine, and hunger ratings. Respiratory quotient was a process measure. Data were analyzed by repeated-measures analysis of variance, with outcomes compared in the early (30 to 150 min) and late (180 to 300 min) postprandial periods.

Results

EA did not differ between the test meals during the early postprandial period (p = 0.99). However, EA in the late postprandial period was significantly lower after the LF test meal than the LGI (p<0.0001) and VLC (p<0.0001) test meals. Metabolic rate also differed in the late postprandial period (p = 0.0074), with higher values on the VLC than LF (p = 0.0064) and LGI (p = 0.0066) diets.

Conclusion

These findings suggest that an LF diet may adversely affect postprandial EA and risk for weight regain during weight loss maintenance.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00315354","term_id":"NCT00315354"}}NCT00315354     

Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells

Fulminant liver failure (FLF) consists of a cascade of events beginning with a presumed uncontrolled systemic activation of the immune system. The etiology of FLF remains undefined. In this study, we demonstrate that CCR5 deficiency promotes the development of acute FLF in mice following Con A administration by preventing activated hepatic CD1d-restricted NKT cells (but not conventional T cells) from dying from activation-induced apoptosis. The resistance of CCR5-deficient NKT cells from activation-induced apoptosis following Con A administration is not due to a defective Fas-driven death pathway. Moreover, FLF in CCR5-deficient mice also correlated with hepatic CCR5-deficient NKT cells, producing more IL-4, but not IFN-gamma, relative to wild-type NKT cells. Furthermore, FLF in these mice was abolished by IL-4 mAb or NK1.1 mAb treatment. We propose that CCR5 deficiency may predispose individuals to the development of FLF by preventing hepatic NKT cell apoptosis and by regulating NKT cell function, establishing a novel role for CCR5 in the development of this catastrophic liver disease that is independent of leukocyte recruitment.