INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

Quantitative Comparison of Ginsenosides and Polyacetylenes in Wild and Cultivated American Ginseng

Quantitative comparison of seven ginsenosides in wild and cultivated American ginseng revealed that the Rg₁/Rd ratio presented a significantly large difference between cultivated and type‐I (one of the defined chemotypes) wild American ginseng, facilitating this ratio as a characteristic marker for differentiating these two groups. Similarly, the ratio (Rg₁+Re)/Rd, and the ratio of protopanaxatriol (PPT)‐type ginsenosides to protopanaxadiol (PPD)‐type ginsenosides showed a large difference between these two groups. On the other hand, type‐II wild samples were found to have high Rg₁/Rb₁ and Rg₁/Re ratios and low panaxydol/panaxynol ratio, which is entirely different from Type‐I American ginseng, but is very similar to that of Asian ginseng. This not only suggests that the chemotype should be taken into consideration properly when using these parameters for differentiating American and Asian ginseng, but also indicates that type‐II wild American ginseng may have distinct pharmacological activities and therapeutic effects.     

Nucleosome linking number change controlled by acetylation of histones H3 and H4

High levels of acetylation of lysines in the amino-terminal domains of all four core histones, H2A, H2B, H3, and H4, have been shown to reduce the linking number change per nucleosome core particle in reconstituted minichromosomes (Norton, V. G., Imai, B. S., Yau, P., and Bradbury, E. M. (1989) Cell 57, 449-457). Because there is evidence to suggest that the acetylations of H3 and H4 have functions that are distinct from those of H2A and H2B, we have determined the nucleosome core particle linking number change in minichromosomes containing fully acetylated H3 and H4 and very low levels of acetylation in H2A and H2B. This linking number change was -0.81 +/- 0.05, in close agreement with the linking number change for hyperacetylated nucleosome core particles which contain high levels of acetylation in all four core histones (approximately 70% of full acetylation in H3 and H4). Therefore, high levels of acetylation of H3 and H4 alone are responsible for the reduction in the linking number change per nucleosome core particle.     

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: I. Kinetics of the Oxidation and Reduction of P-870 As Affected by External Factors

Photosynthetic reaction centers from Rhodopseudomonas spheroides were prepared with the detergent lauryl dimethylamine oxide (LDAO). In contrast to reaction centers made with Triton X-100, these contained no cytochromes and little or no ubiquinone (UQ). The reduction of P-870, after its photochemical oxidation, was studied in these materials with the following results. In reaction centers made with Triton X-100, slow kinetic components (seconds to minutes) could be attributed to secondary electron acceptors or traps. In reaction centers made with LDAO the kinetics were predominantly fast (half-times, 100 msec or less); slower components could be introduced by adding UQ. Added UQ appeared to become bound to reaction centers made with LDAO, but the binding might have meant only that both components were trapped within detergent micelles. Ferricyanide could retard the reduction of oxidized P-870, apparently by capturing electrons from the reducing side of the photochemical system. Under conditions in which the participation of secondary electron acceptors seemed to have been eliminated, the recovery of P-870 was mainly by a first-order process with a half-time of about 60 msec at room temperature and 20-30 msec at about -80°C and below. The transition with decreasing temperature suggested the presence of a mixed population, exhibiting both the 60 and 20 msec components, but variations in the absorption spectra with temperature did not suggest the presence of a mixed population. Absorption difference spectra in the ultraviolet were compatible with the idea that UQ added to reaction centers became reduced in the light.     

Field-effect amperometric immuno-detection of protein biomarker

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work.     

Aging impairs human bone marrow function and cardiac repair following myocardial infarction in a humanized chimeric mouse

Ventricular remodeling following myocardial infarction (MI) is a major cause of heart failure, a condition prevalent in older individuals. Following MI, immune cells are mobilized to the myocardium from peripheral lymphoid organs and play an active role in orchestrating repair. While the effect of aging on mouse bone marrow (BM) has been studied, less is known about how aging affects human BM cells and their ability to regulate repair processes. In this study, we investigate the effect aging has on human BM cell responses post‐MI using a humanized chimeric mouse model. BM samples were collected from middle aged (mean age 56.4 ± 0.97) and old (mean age 72.7 ± 0.59) patients undergoing cardiac surgery, CD34^+/− cells were isolated, and NOD‐scid‐IL2rγ^null (NSG) mice were reconstituted. Three months following reconstitution, the animals were examined at baseline or subjected to coronary artery ligation (MI). Younger patient cells exhibited greater repopulation capacity in the BM, blood, and spleen as well as greater lymphoid cell production. Following MI, CD34⁺ cell age impacted donor and host cellular responses. Mice reconstituted with younger CD34⁺ cells exhibited greater human CD45⁺ recruitment to the heart compared to mice reconstituted with old cells. Increased cellular responses were primarily driven by T‐cell recruitment, and these changes corresponded with greater human IFNy levels and reduced mouse IL‐1β in the heart. Age‐dependent changes in BM function led to significantly lower survival, increased infarct expansion, impaired host cell responses, and reduced function by 4w post‐MI. In contrast, younger CD34⁺ cells helped to limit remodeling and preserve function post‐MI.     

Length‐weight relationships of 79 marine fish species from the coastal waters of Hong Kong

Dedicated benthic fisheries‐assessment surveys with beam trawls (2 cm mesh size) were used to sample demersal fish communities at depths 7–35 m along four transects in each of the western, southern and eastern waters of Hong Kong monthly for >24 months. We report length‐weight relationships (LWRs) for 79 species (11 orders, 37 families) of fishes for which LWRs are presently absent or scarce. For 56 of these species, relationships between total length and standard length or disc width were also derived.     

Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells

A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. "brown kidney bean." The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20 °C-60 °C. However, drastic reduction of the activity occurred at temperatures above 65 °C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0-2, and only residual activity was observed at pH 13-14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 μM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC(50) of 5.12 μM, 32.85 μM, 3.12 μM and 40.12 μM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.     

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.