Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation

B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI and BAFF-R. We have determined the crystal structure of the extracellular domain of BAFF-R bound to BAFF at a resolution of 3.3 A. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a beta-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the beta-turn and is indispensable in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. The extracellular domain of TACI contains two CRDs, both of which contain the DxL motif. Modeling of TACI-BAFF complex suggests that both CDRs simultaneously interact with the BAFF dimer in the virus-like cage.     

A data review and re-assessment of ovarian cancer serum proteomic profiling

Background

The early detection of ovarian cancer has the potential to dramatically reduce mortality. Recently, the use of mass spectrometry to develop profiles of patient serum proteins, combined with advanced data mining algorithms has been reported as a promising method to achieve this goal. In this report, we analyze the Ovarian Dataset 8-7-02 downloaded from the Clinical Proteomics Program Databank website, using nonparametric statistics and stepwise discriminant analysis to develop rules to diagnose patients, as well as to understand general patterns in the data that may guide future research.

Results

The mass spectrometry serum profiles derived from cancer and controls exhibited numerous statistical differences. For example, use of the Wilcoxon test in comparing the intensity at each of the 15,154 mass to charge (M/Z) values between the cancer and controls, resulted in the detection of 3,591 M/Z values whose intensities differed by a p-value of 10^-6 or less. The region containing the M/Z values of greatest statistical difference between cancer and controls occurred at M/Z values less than 500. For example the M/Z values of 2.7921478 and 245.53704 could be used to significantly separate the cancer from control groups. Three other sets of M/Z values were developed using a training set that could distinguish between cancer and control subjects in a test set with 100% sensitivity and specificity.

Conclusion

The ability to discriminate between cancer and control subjects based on the M/Z values of 2.7921478 and 245.53704 reveals the existence of a significant non-biologic experimental bias between these two groups. This bias may invalidate attempts to use this dataset to find patterns of reproducible diagnostic value. To minimize false discovery, results using mass spectrometry and data mining algorithms should be carefully reviewed and benchmarked with routine statistical methods.

     

Lipopolysaccharide enhances the cytotoxicity of 2-chloroethyl ethyl sulfide

Background

The bacterial endotoxin, lipopolysaccharide (LPS), is a well-characterized inflammatory factor found in the cell wall of Gram-negative bacteria. In this investigation, we studied the cytotoxic interaction between 2-chloroethyl ethyl sulfide (CEES or ClCH₂CH₂SCH₂CH₃) and LPS using murine RAW264.7 macrophages. CEES is a sulfur vesicating agent and is an analog of 2,2'-dichlorodiethyl sulfide (sulfur mustard). LPS is a ubiquitous natural agent found in the environment. The ability of LPS and other inflammatory agents (such as TNF-alpha and IL-1beta) to modulate the toxicity of CEES is likely to be an important factor in the design of effective treatments.

Results

RAW 264.7 macrophages stimulated with LPS were found to be more susceptible to the cytotoxic effect of CEES than unstimulated macrophages. Very low levels of LPS (20 ng/ml) dramatically enhanced the toxicity of CEES at concentrations greater than 400 μM. The cytotoxic interaction between LPS and CEES reached a maximum 12 hours after exposure. In addition, we found that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) as well as phorbol myristate acetate (PMA) also enhanced the cytotoxic effects of CEES but to a lesser extent than LPS.

Conclusion

Our in vitro results suggest the possibility that LPS and inflammatory cytokines could enhance the toxicity of sulfur mustard. Since LPS is a ubiquitous agent in the natural environment, its presence is likely to be an important variable influencing the cytotoxicity of sulfur mustard toxicity. We have initiated further experiments to determine the molecular mechanism whereby the inflammatory process influences sulfur mustard cytotoxicity.

     

遗传学实验（十五）两倍体细胞株培养

实验原理 组织培养是把动物或植物细胞自机体取出放在玻 璃器皿里,选择和控制某些外界条件,使细胞继续分裂 生长的一种基础性实验技术;现在已广泛应用于生理 学、免疫学、病毒学、遗传学等方面,对细胞分化、发育、 肿瘤发生以及染色体研究等领域起着很大的作用。     

Transmodulation between phospholipase D and c-Src enhances cell proliferation

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.     

Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 are essential for sister chromatid cohesion in meiosis and mitosis

Sister chromatid cohesion is fundamental for the faithful transmission of chromosomes during both meiosis and mitosis. Proteins involved in this process are highly conserved from yeasts to humans. In screenings for sterile animals with abnormal vulval morphology, mutations in the Caenorhabditis elegans evl-14 and scc-3 genes were isolated. Defects in cell divisions were observed in germ line as well as in vulval and somatic gonad lineages. Through positional cloning of these genes, we have shown that EVL-14 and SCC-3 are likely the only C. elegans homologs of the yeast sister chromatid cohesion proteins Pds5 and Scc3, respectively. Both evl-14 and scc-3 mutants displayed defects in the meiotic germ line. In evl-14 mutants, synaptonemal complexes (SCs) were detectable but more than the usual six DAPI (4',6'-diamidino-2-phenylindole)-positive structures were seen at diakinesis, suggesting that EVL-14/PDS-5 is important for the maintenance of sister chromatid cohesion in late prophase. In scc-3 mutant animals, normal SCs were not visible and approximately 24 DAPI-positive structures were seen at diakinesis, indicating that SCC-3 is necessary for sister chromatid cohesion. Immunostaining revealed that localization of REC-8, a homolog of the yeast meiotic cohesin subunit Rec8, to the chromosomes depends on the presence of SCC-3 but not that of EVL-14/PDS-5. scc-3 RNA interference (RNAi)-treated embryos were 100% lethal and displayed defects in cell divisions. evl-14 RNAi caused a range of phenotypes. These results indicate that EVL-14/PDS-5 and SCC-3 have functions in both mitosis and meiosis.     

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg(2+), Ca(2+), Sr(2+) and Ba(2+) were used for clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg(2+), Ca(2+) and Sr(2+) are counterions. When Ba(2+) is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B-A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr(2+) induces the greatest structural transition.     

Region-specific and stage-dependent regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 homeodomain transcription factor

During early neural development, the Nkx6.1 homeodomain neural progenitor gene is specifically expressed in the ventral neural tube, and its activity is required for motoneuron generation in the spinal cord. We report that Nkx6.1 also controls oligodendrocyte development in the developing spinal cord, possibly by regulating Olig gene expression in the ventral neuroepithelium. In Nkx6.1 mutant spinal cords, expression of Olig2 in the motoneuron progenitor domain is diminished, and the generation and differentiation of oligodendrocytes are significantly delayed and reduced. The regulation of Olig gene expression by Nkx6.1 is stage dependent, as ectopic expression of Nkx6.1 in embryonic chicken spinal cord results in an induction of Olig2 expression at early stages, but an inhibition at later stages. Moreover, the regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 also appears to be region specific. In the hindbrain, unlike in the spinal cord, Olig1 and Olig2 can be expressed both inside and outside the Nkx6.1-expressing domains and oligodendrogenesis in this region is not dependent on Nkx6.1 activity.     

ADP is produced by firefly luciferase but its synthesis is independent of the light emitting properties

During experiments aimed at understanding the time course of appearance of reaction products in the Photinus pyralis luciferase system, an expected compound with a typical nucleotide UV spectrum was isolated. According to capillary electrophoresis (CE) analysis and 1H, 31P-NMR spectra, it was unambiguously found to be ADP, either with extracted or recombinant enzymes. The ADP synthesis was demonstrated by standard UV spectrophotometric methods and CE analysis. Also, the luciferase produced AMP and ATP from ADP. This reaction was completely inhibited by Ap(5)A at 250 nM and was independent of the light emitting properties of the enzyme. The only catalytic mechanism to explain the production of ADP is an intrinsic adenylate kinase activity of luciferase. The K(m) values of the AK activity are 0.3, 0.7, 0.06 mM for AMP, ADP, and ATP respectively. The multiple enzyme activities of luciferase may be partly responsible for the complex kinetics of light emission by changing the nucleotide concentrations.