Polyene substrates with unusual methylation patterns to probe the active sites of three catalytic antibodies

The synthesis of two tetraenes that differ in their methylation pattern from the natural substrate in lanosterol biosynthesis, 2,3-oxidosqualene, and their examination with three catalytic antibodies is described. The design of these novel, linear terpenoid structures was governed by initial results obtained from the characterization of the three catalytic antibodies. These were generated by immunization with a steroidal hapten that mimics multicyclization without the necessity for anti-Markovnikov additions or ring expansions. Such a reaction cascade would represent a more 'primitive' version compared to the oxidosqualene cyclization observed in lanosterol, cycloartenol and beta-amyrin biosynthesis and would not require a tail-to-tail connection of the third and fourth isoprene unit as seen in squalene. The first tetraene design (A) only contains trisubstituted double bonds and hence its synthesis starts from farnesol and tris-norgeraniol. The second tetraene design (B) is considered the more precise match to the inducing hapten that generated the antibody collections by exhibiting one disubstituted double bond and its synthesis utilizes a tris-norgeraniol derivative and a symmetrical bis-allylic alcohol as key building blocks. Chromatographic comparison studies lead to the conclusion that the currently studied antibodies also produce monocyclic products from the two substrates as has been formerly observed with a squalene-derived substrate. In contrast, 2,3-oxidosqualene is not accepted by these catalysts supporting the notion that the current substrates are fully bound by recognition of both terminal functional groups.     

Photosynthetic Characterization of the Plant Dicranopteris dichotoma Bernh. in a Rare Earth Elements Mine

In order to investigate the distribution of rare earth elements （REEs） in the natural hyperaccumulator fern Dicranopteris dichotoma Bernh. and to characterize this plant photosynthetically, concentrations of REEs in D. dichotoma from mines mining heavy and light REEs （HREEs and LREEs, respectively）, as well as in D. dichotoma from an area in which no mining occurred, in southern Jiangxi Province were determined using inductively coupled plasma-mass spectrometry. The REE concentrations in the lamina of D. dichotoma were in the order LREEs mine 〉 HREEs mine 〉 non-mining area. The maximum REE content in the lamina of D. dichotoma from the LREE mine was approximately 2 648 mg/kg dry weight. The photosynthetic activity of D. dichotoma from areas of HREE and LREE mines was improved by the presence of high concentrations of REEs in the lamina compared with D. dichotoma from the non-mining area. However, this enhancement varied according to the concentrations of the REEs, as well as their type. In addition, 77K fluorescence, electron transport rate, and chlorophyll-protein complex studies showed that the enhancement of the photosynthetic activity of D. dichotoma from HREE mines was mainly due to an increase in the chlorophyll-protein complex of the reaction center of photosystem （PS） Ⅰ, whereas the enhancement observed in D. dichotoma from LREE mines was due to an increase in the internal antennae chlorophyll-protein complex of PS Ⅱ and greater light energy distribution to the light-harvesting chlorophyll-protein complex of PS Ⅱ.     

A New Isoflavonoid from Belamcanda chinensis （L.） DC.

A new isoflavonoid, 5, 6, 7, 3＇-terahydroxy-8, 4＇, 5＇-trimethoxyisoflavone （1）, along with 10 known isoflavonoids, namely 5, 6, 7, 4＇-tetrahydroxy-8-methoxyisoflavone （2）, irilone （3）, genistein （4）, tectorigenin （5）, irigenin （6）, irisflorentin （7）, dichotomitin （8）, dimethyltectorigenin （9）, iridin （10）, and tectoridin （11）, was isolated from the alcohol extract of the rhizomes of Belamcanda chinensis （L.） DC. The structures of these compounds were elucidated on the basis of results of spectroscopic analysis.     

Deep membrane insertion of prion protein upon reduction of disulfide bond

The membrane may play a role in the pathogenesis of the prion protein (PrP). Cytoplasmic expression of PrP causes the conversion of PrP to a self-perpetuating PrP^Sc-like conformation and the interaction of polypeptide chain with the hydrophobic core of the membrane is believed to be closely correlated with neurodegeneration. However, it is still elusive what factors govern the membrane interaction of PrP. Here, we show that PrP penetrates deeply into the membrane when the single disulfide bond is reduced, which results in membrane disruption and leakage. The proteinase K treatment and the fluorescence quenching assays showed that a predicted transmembrane domain of PrP penetrates into the membrane when the disulfide bond was reduced. Therefore, the oxidation state of PrP might be an important factor that influences its neurotoxicity or pathogenesis.     

Stress hormones and immune function

Over the past 20 years we have demonstrated both in animal models and in human studies that stress increases neuroendocrine hormones, particularly glucocorticoids and catecholamines but to some extent also prolactin, growth hormone and nerve growth factor. We have also shown that stress, through the action of these stress hormones, has detrimental effects on immune function, including reduced NK cell activity, lymphocyte populations, lymphocyte proliferation, antibody production and reactivation of latent viral infections. Such effects on the immune system have severe consequences on health which include, but are not limited to, delayed wound healing, impaired responses to vaccination and development and progression of cancer. These data provide scientific evidence of the effects of stress on immune function and implications for health.     

Central role of reverting mutations in HLA associations with human immunodeficiency virus set point

Much uncertainty still exists over what T-cell responses need to be induced by an effective human immunodeficiency virus (HIV) vaccine. Previous studies have hypothesized that the effective CD8⁺ T-cell responses are those driving the selection of escape mutations that reduce viral fitness and therefore revert posttransmission. In this study, we adopted a novel approach to define better the role of reverting escape mutations in immune control of HIV infection. This analysis of sequences from 710 study subjects with chronic C-clade HIV type 1 infection demonstrates the importance of mutations that impose a fitness cost in the control of viremia. Consistent with previous studies, the viral set points associated with each HLA-B allele are strongly correlated with the number of Gag-specific polymorphisms associated with the relevant HLA-B allele (r = −0.56, P = 0.0034). The viral set points associated with each HLA-C allele were also strongly correlated with the number of Pol-specific polymorphisms associated with the relevant HLA-C allele (r = −0.67, P = 0.0047). However, critically, both these correlations were dependent solely on the polymorphisms identified as reverting. Therefore, despite the inevitable evolution of viral escape, viremia can be controlled through the selection of mutations that are detrimental to viral fitness. The significance of these results is in highlighting the rationale for an HIV vaccine that can induce these broad responses.     

Effect of metabolic inhibition on couplon behavior in rabbit ventricular myocytes

We investigated the effect of combined inhibition of oxidative and glycolytic metabolism on L-type Ca²⁺ channels (LCCs) and Ca²⁺ spikes in isolated patch-clamped rabbit ventricular myocytes. Metabolic inhibition (MI) reduced LCC open probability, increased null probability, increased first latency, and decreased open time but left conductance unchanged. These results explain the reduction in macroscopic Ca²⁺ current observed during MI. MI also produced a gradual reduction in action potential duration at 90% repolarization (APD₉₀), a clear decline in spike probability, and an increase in spike latency and variance. These effects are consistent with the changes we observed in LCC activity. MI had no effect on the amplitude or time to peak of Ca²⁺ spikes until APD₉₀ reached 10% of control, suggesting preserved sarcoplasmic reticulum Ca²⁺ stores and ryanodine receptor (RyR) conductance in those couplons that remained functioning. Ca²⁺ spikes disappeared completely when APD₉₀ reached <2% of control, although in two cells, spikes were reactivated in a highly synchronized fashion by very short action potentials. This reactivation is probably due to the increased driving force for Ca²⁺ entry through a reduced number of LCCs that remain open during early repolarization. The enlarged single channel flux produced by rapid repolarization is apparently sufficient to trigger RyRs whose Ca²⁺ sensitivity is likely reduced by MI. We suggest that loss of coupling fidelity during MI is explained by loss of LCC activity (possibly mediated by Ca²⁺-calmodulin kinase II activity). In addition, the results are consistent with loss of RyR activity, which can be mitigated under conditions likely to enlarge the trigger.     

Novel features of the rabbit transverse tubular system revealed by quantitative analysis of three-dimensional reconstructions from confocal images

With scanning confocal microscopy we obtained three-dimensional (3D) reconstructions of the transverse tubular system (t-system) of rabbit ventricular cells. We accomplished this by labeling the t-system with dextran linked to fluorescein or, alternatively, wheat-germ agglutinin conjugated to an Alexa fluor dye. Image processing and visualization techniques allowed us to reconstruct the t-system in three dimensions. In a myocyte lying flat on a coverslip, t-tubules typically progressed from its upper and lower surfaces. 3D reconstructions of the t-tubules also suggested that some of them progressed from the sides of the cell. The analysis of single t-tubules revealed novel morphological features. The average diameter of single t-tubules from six cells was estimated to 448 ± 172 nm (mean ± SD, number of t-tubules 348, number of cross sections 5323). From reconstructions we were able to identify constrictions occurring every 1.87 ± 1.09 μm along the principal axis of the tubule. The cross-sectional area of these constrictions was reduced to an average of 57.7 ± 27.5% (number of constrictions 170) of the adjacent local maximal areas. Principal component analysis revealed flattening of t-tubular cross sections, confirming findings that we obtained from electron micrographs. Dextran- and wheat-germ agglutinin-associated signals were correlated in the t-system and are therefore equally good markers. The 3D structure of the t-system in rabbit ventricular myocytes seems to be less complex than that found in rat. Moreover, we found that t-tubules in rabbit have approximately twice the diameter of those in rat. We speculate that the constrictions (or regions between them) are sites of dyadic clefts and therefore can provide geometric markers for colocalizing dyadic proteins. In consideration of the resolution of the imaging system, we suggest that our methods permit us to obtain spatially resolved 3D reconstructions of the t-system in rabbit cells. We also propose that our methods allow us to characterize pathological defects of the t-system, e.g., its remodeling as a result of heart failure.     

Effects of Bt transgenic Chinese cabbage on the herbivore Mamestra brassicae (Lepidoptera: Noctuidae) and its parasitoid Microplitis mediator (Hymenoptera: Braconidae)

We investigated the effects of a diamondback moth-resistant Chinese cabbage (Brassica campestris subsp napus variety pekinensis Makino), expressing the insecticidal protein CrylA(c) toxin derived from Bacillus thuringiensis, on the nontarget herbivore Mamestra brassicae (L.) (Lepidoptera: Noctuidae) and its parasitoid wasp Microplitis mediator (Haliday) (Hymenoptera: Braconidae). A decreased survival rate at neonate stage was observed in M. brassicae when reared on Bt cabbage, although overall development was not significantly affected. According to enzyme-linked immunosorbent assay test using CrylA(c) antibody, the Cry toxin was only detected in the alimentary canal, not in the hemolymph or remaining body parts of M. brassicae, indicating that the ingested Cry toxin is neither distributed inside the body nor transferred through the trophic level. As expected, no Cry toxin was found in the larvae and cocoons of M. mediator. In addition, no significant changes were observed in the parasitization rate, larval period, pupal period, cocoon weight, or adult emergence rate when M. mediator wasps were reared on the M. brassicae larvae fed with transgenic Chinese cabbage. In summary, no direct or indirect adverse effects of transgenic Chinese cabbage on the two nontarget insect species were observed, suggestive of low risk in herbivore-parasitoid food chain.     

Increased frequency of EBV-specific effector memory CD8+ T cells correlates with higher viral load in rheumatoid arthritis

EBV is a candidate trigger of rheumatoid arthritis (RA). We determined both EBV-specific T cell and B cell responses and cell-associated EBV DNA copies in patients with RA and demographically matched healthy virus carriers. Patients with RA showed increased and broadened IgG responses to lytic and latent EBV-encoded Ags and 7-fold higher levels of EBV copy numbers in circulating blood cells. Additionally, patients with RA exhibited substantial expansions of CD8(+) T cells specific for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8(+) T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4(+) T cell responses to EBV and T cell responses to human CMV Ags were unchanged, altogether arguing against a defective control of latent EBV infection in RA. Our data show that the regulation of EBV infection is perturbed in RA and suggest that increased EBV-specific effector T cell and Ab responses are driven by an elevated EBV load in RA.