Search for Light Weakly-Interacting-Massive-Particle Dark Matter by Annual Modulation Analysis with a Point-Contact Germanium Detector at the China Jinping Underground Laboratory.

We present results on light weakly interacting massive particle (WIMP) searches with annual modulation (AM) analysis on data from a 1-kg mass p-type point-contact germanium detector of the CDEX-1B experiment at the China Jinping Underground Laboratory. Datasets with a total live time of 3.2 yr within a 4.2-yr span are analyzed with analysis threshold of 250 eVee. Limits on WIMP-nucleus (χ-N) spin-independent cross sections as function of WIMP mass (m_{χ}) at 90% confidence level (C.L.) are derived using the dark matter halo model. Within the context of the standard halo model, the 90% C.L. allowed regions implied by the DAMA/LIBRA and CoGeNT AM-based analysis are excluded at >99.99% and 98% C.L., respectively. These results correspond to the best sensitivity at m_{χ}<6 GeV/c^{2} among WIMP AM measurements to date.

Constraints on Spin-Independent Nucleus Scattering with sub-GeV Weakly Interacting Massive Particle Dark Matter from the CDEX-1B Experiment at the China Jinping Underground Laboratory.

We report results on the searches of weakly interacting massive particles (WIMPs) with sub-GeV masses (m_{χ}) via WIMP-nucleus spin-independent scattering with Migdal effect incorporated. Analysis on time-integrated (TI) and annual modulation (AM) effects on CDEX-1B data are performed, with 737.1 kg day exposure and 160 eVee threshold for TI analysis, and 1107.5 kg day exposure and 250 eVee threshold for AM analysis. The sensitive windows in m_{χ} are expanded by an order of magnitude to lower DM masses with Migdal effect incorporated. New limits on σ_{χN}^{SI} at 90% confidence level are derived as 2×10^{-32}∼7×10^{-35} cm^{2} for TI analysis at m_{χ}∼50-180 MeV/c^{2}, and 3×10^{-32}∼9×10^{-38} cm^{2} for AM analysis at m_{χ}∼75 MeV/c^{2}-3.0 GeV/c^{2}.

Limits on Light Weakly Interacting Massive Particles from the First 102.8 kg×day Data of the CDEX-10 Experiment.

We report the first results of a light weakly interacting massive particles (WIMPs) search from the CDEX-10 experiment with a 10 kg germanium detector array immersed in liquid nitrogen at the China Jinping Underground Laboratory with a physics data size of 102.8 kg day. At an analysis threshold of 160 eVee, improved limits of 8×10^{-42} and 3×10^{-36} cm^{2} at a 90% confidence level on spin-independent and spin-dependent WIMP-nucleon cross sections, respectively, at a WIMP mass (m_{χ}) of 5 GeV/c^{2} are achieved. The lower reach of m_{χ} is extended to 2 GeV/c^{2}.

Oxidation of human lens recombinant alphaA-crystallin and cysteine-deficient mutants.

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H202 oxidation in an ascorbate-FeCl(3)-EDTA-H202 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type alphaA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H202. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.

Spectral contribution of the individual tryptophan of alphaB-crystallin: a study by site-directed mutagenesis.

There are two tryptophan residues in the lens alphaB-crystallin, Trp9 and Trp60. We prepared two Trp --> Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near-ultraviolet circular dichroism (UV CD) differently. The major difference in the near-UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near-UV CD shows an increased intensity in the region of 270-280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a beta-strand.

Subunit exchange of lens alpha-crystallin: a fluorescence energy transfer study with the fluorescent labeled alphaA-crystallin mutant W9F as a probe.

A Trp-free alphaA-crystallin mutant (W9F) was prepared by site-directed mutation. This mutant appears to be identical to the wild-type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl-specific fluorescent probe, 2-(4'-maleimidylanilino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange between alphaA- and alphaA-crystallins as well as between alphaA- and alphaB-crystallins, studied by measurement of fluorescence resonance energy transfer. Energy transfer was observed between Trp (donor, with emission maximum at 336 nm) of wild-type alphaA- or alphaB-crystallin and MIANS (acceptor, with absorption maximum at 313 nm) of labeled W9F when subunit exchange occurred. Time-dependent decrease of Trp and increase of MIANS fluorescence were recorded. The exchange was faster at 37 degrees C than at 25 degrees C. The energy transfer efficiency was greater between homogeneous subunits (alphaA-alphaA) than between heterogeneous subunits (alphaA-alphaB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between alphaA and alphaB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between alphaA-alphaA and alphaA-alphaB indicated that alphaA- and alphaB-crystallins were not necessarily structurally equivalent.

Fluorescence and immunochemical studies of advanced glycation-related lens pigments.

PURPOSE: To establish whether advanced glycation is the major mechanism for yellowing of lens proteins. METHODS: Synchronous fluorescence (SF) and immunochemical assays were used to study glycation in vitro and in vivo. In the in vitro study, advanced glycation end products (AGEs) were prepared and used as antigens to induce antibodies to AGEs. The in vitro AGEs and classified nuclear cataracts were analyzed by SF and immunochemical assays. RESULTS: In vitro AGEs generated from various glycating agents and carrier proteins displayed strong SF above 350 nm; the spectra were well resolved with major bands at 380 nm and 420 nm. Samples from human lenses manifested a band at 395 nm in addition to the two bands shown by in vitro AGEs. SF intensity is greater for the water-insoluble (WI) than water-soluble (WS) fraction, but both increased with increasing nuclear color. The immunoreactivity data also showed that the WI fraction contained more AGEs than the WS fraction and that the amount of AGEs increased with increasing nuclear color. CONCLUSIONS: Fluorescence and immunoassays indicated that pigmented AGEs contributed to yellowing of the crystalline lens nucleus.

Heat-induced conformational change of human lens recombinant alphaA- and alphaB-crystallins.

PURPOSE: To determine which component of lens alpha-crystallin is responsible for heat-induced transition, conformational change and high molecular weight (HMW) aggregation. METHODS: Recombinant alphaA- and alphaB-crystallins were used. Temperature dependent changes were probed by Trp fluorescence and circular dichroism (CD) measurements. HMW aggregates were induced by heating at 62 degrees C for 1-2 h and then cooling to room temperature. The nature of HMW aggregation was studied with fluorescent probes, 4,4'-dianilino-1, 1'-binaphthalene-5,5'-disulfonic acid (bis-ANS) and thioflavin T (ThT). RESULTS: CD and Trp fluorescence revealed that alphaB-crystallin was more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation. At temperatures greater than 70 degrees C, alphaB-crystallin precipitated but alphaA-crystallin remained soluble. Both bis-ANS and ThT probes displayed increased fluorescence intensity with HMW aggregation, but the increase for bis-ANS was greater with alphaB-crystallin than with alphaA-crystallin, while the reverse was true for ThT. CONCLUSIONS: These results indicate that alphaB-crystallin is more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation and are consistent with the notion that alphaA- and alphaB-crystallins have different biochemical and biophysical properties in spite of their high degree of homology.

Photooxidized products of recombinant alpha A-crystallin and W9F mutant.

Human lenses contain many photosensitizers that absorb light at wavelengths above 300 nm, most notably UVA light (320-400 nm). Kynurenine (Kyn) and 3-hydroxykynurenine (HK), two of the best-known photosensitizers in the human lens, may play a significant role in photooxidation-related changes in lens proteins, such as conformational change and aggregation. In vitro irradiation experiments with proteins indicate that the Trp residue (with maximal absorption at 295 nm) is more susceptible to photooxidation by UVB light (280-320 nm) than by UVA light, but most UVB light below 300 nm is screened by the cornea and little reaches the lens, especially the nuclear region where nuclear color develops. Therefore, if photooxidation is an important contributor to nuclear color or nuclear cataract, it must arise from a photosensitized reaction. In the present study, we use recombinant alpha A- and its Trp-deficient mutant W9F as models to study the effects of UVA irradiation in the presence of HK or Kyn and of UVB (300 nm) irradiation on alpha-crystallins. alpha A-crystallin showed a large decrease in Trp fluorescence and a large increase in non-Trp (blue) fluorescence after the HK-sensitized or 300 nm photooxidation. For the W9F mutant, a smaller decrease in protein fluorescence (lambda ex at 280 nm) and a smaller increase in blue fluorescence than for the wild-type alpha A-crystallin were observed. A decrease in the near-UV CD was also observed for both photooxidized alpha A and the W9F mutant. The effect of Kyn sensitization is smaller than that of HK sensitization. A study of chaperone-like activity indicated that only 300 nm photooxidized alpha A and the W9F mutant increased the ability to protect insulin from dithiothreitol-induced aggregation. Thus, sensitized photooxidation can occur in amino acids other than Trp by UVA in the presence of HK or Kyn with effects similar to, albeit smaller than, those of direct UVB (300 nm) photooxidation.

Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant alpha-crystallins.

PURPOSE: Lens proteins underwent nonenzymatic glycation, and the advanced glycation end products (AGEs) were detected by immunological assays. One of the major AGE structures is N(epsilon)-(carboxymethyl)lysine (CML). Since the involvement of AGEs in the pathogenesis of diabetic complications is speculated, the effects of CML formation on proteins were studied. METHODS: CML adducts were generated in recombinant alphaA- and alphaB-crystallins by incubation with glyoxylic acid and NaBH3CN. SDS-PAGE and size exclusion chromatography were used to detect subunit degradation and high-molecular-weight (HMW) aggregation. Conformational change was determined by fluorescence and circular dichroism (CD) measurements. The chaperone function was studied by DTT-induced aggregation of insulin. RESULTS: Lysine modification was estimated to be 60-90% depending on the conditions of incubation. No subunit degradation or HMW aggregation was observed. Fluorescence and CD measurements detected a conformational change in CML adducts. Measurements of chaperone-like activity, however, indicated that the formation of CML increased the protein's ability to protect insulin against DTT-induced aggregation. CONCLUSIONS: Although CML adducts of alphaA- and alphaB-crystallins, the major AGE structures formed in vitro, changed protein conformation, no subunit degradation and HMW aggregation were observed. Moreover, the CML adducts increased chaperone-like activity of both alphaA- and alphaB-crystallins. The results suggest that CML formation alone may not play a major role in protein aggregation and lens opacity.

Combinatorial search for advanced luminescence materials.

Phosphors are key materials in fluorescent lighting, displays, x-ray scintillation, etc. The rapid development of modern photonic technologies, e.g., mercury-free lamps, flat panel displays, CT-detector array, etc., demands timely discovery of advanced phosphors. To this end, a combinatorial approach has been developed and applied to accelerated experimental search of advanced phosphors and scintillators. Phosphor libraries can be made in both thin film and powder form, using masking strategies and liquid dispensing systems, respectively. High-density libraries with 100 to 1000 discrete phosphor compositions on a 1"-square substrate can be made routinely. Both compositions and synthesis temperatures can be screened in a high-throughput mode. In this article, details on the existing methods of combinatorial synthesis and screening of phosphors will be reported with examples. These methods are generic tools for application of combinatorial chemistry in the discovery of other solid state materials. A few highly efficient phosphors discovered with combinatorial methods have been reproduced in bulk form and their luminescent properties measured.

Intermolecular exchange and stabilization of recombinant human alphaA- and alphaB-crystallin.

Lens alpha-crystallin subunits alphaA and alphaB are differentially expressed and have a 3-to-1 ratio in most mammalian lenses by intermolecular exchange. The biological significance of this composition and the mechanism of exchange are not clear. Preparations of human recombinant alphaA- and alphaB-crystallins provide a good system in which to study this phenomenon. Both recombinant alphaA- and alphaB-crystallins are folded and aggregated to the size of the native alpha-crystallin. During incubation together, they undergo an intermolecular exchange as shown by native isoelectric focusing. Circular dichroism measurements indicate that the protein with a 3-to-1 ratio of alphaA- and alphaB-crystallins has the same secondary structure but somewhat different tertiary structures after exchange: the near-UV CD increases after exchange. The resulting hybrid aggregate is more stable than the individual homogeneous aggregates: at 62 degrees C, alphaB-crystallin is more susceptible to aggregation and displays a greater light scattering than alphaA-crystallin. This heat-induced aggregation of alphaB-crystallin, however, was suppressed by intermolecular exchange with alphaA-crystallin. These phenomena are also observed by fast performance liquid chromatography gel filtration patterns. The protein structure of alphaB-crystallin is stabilized by intermolecular exchange with alphaA-crystallin.

Conformational and functional differences between recombinant human lens alphaA- and alphaB-crystallin.

Human and other mammalian lens proteins are composed of three major crystallins: alpha-, beta-, and gamma-crystallin. alpha-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency. With age, the crystallins, especially alpha-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation. Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments. alpha-Crystallin is composed of two subunits, alphaA and alphaB. Before the application of recombinant DNA technology, these two alpha-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly. In the present studies, we applied the recombinant DNA technology to prepare native, unmodified alphaA- and alphaB-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, alphaA and alphaB cDNAs were isolated from a human lens epithelial cell cDNA library. The cDNAs were cloned into a pAED4 expression vector and then expressed in E. coli strain BL21(DE3). Pure recombinant alphaA- and alphaB-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography. They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity. alphaA- and alphaB-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that alphaB-crystallin is more hydrophobic than alphaA-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for alphaB- than for alphaA-crystallin. The resulting data provide a base line for further studies of human lens alpha-crystallin.

Conformational change of human lens insoluble alpha-crystallin.

Human lens alpha-crystallin becomes progressively insoluble with age and is the major crystallin component in the water-insoluble (WI) fraction. The mechanism that causes the originally water-soluble (WS) alpha-crystallin to become insoluble is unknown. A conformational change by chemical modification may be the cause, but the nature of insolubility renders it impossible to study protein conformation in the WI fraction by most spectroscopic measurements. In the present study, alpha-crystallin in the WI fraction was extracted by urea and reconstituted to a folded protein by dialysis. The refolded urea-soluble (US) alpha-crystallin was compared with WS alpha-crystallin. The US alpha-crystallin has a greater amount of polymeric species, but fewer degraded subunits than the WS alpha-crystallin as shown by SDS-PAGE and Western blot. Circular dichroism (CD) measurements indicate that they have the same secondary structure but a different tertiary structure, possibly a partial unfolding in the US alpha-crystallin. This is supported by fluorescence measurements: Trp residues are more exposed and protein has a more-hydrophobic surface in the US than in the WS alpha-crystallin. Blue fluorescence further indicates that the US alpha-crystallin has a greater amount of pigment than the WS alpha-crystallin. Together, these results indicate that the US alpha-crystallin is a chemically and conformationally modified protein.

Thermodynamic stability of human lens recombinant alphaA- and alphaB-crystallins.

Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules.

Renal concentrating defect in mice lacking group IV cytosolic phospholipase A(2).

Eicosanoids regulate various cellular functions that are important in physiological and pathophysiological processes. Arachidonic acid is released from membranes by phospholipase A(2) (PLA(2)) activity. Activated macrophages derived from mice lacking the 85-kDa group IV cytosolic PLA(2) (cPLA(2)) have a markedly reduced release of prostaglandin E(2) and leukotrienes B(4) and C(4). Under basal conditions and after furosemide, urinary prostaglandin E(2) excretion is reduced in cPLA(2)-knockout (cPLA(2)(-/-)) mice. Serum creatinine, Na(+), K(+), and Ca(2+) concentrations, glomerular filtration rate, and fractional excretion of Na(+) and K(+) are not different in cPLA(2)(-/-) and cPLA(2)(+/+) mice. Maximal urinary concentration is lower in 48-h water-deprived cPLA(2)(-/-) mice compared with cPLA(2)(+/+) animals (1,934 +/- 324 vs. 3,541 +/- 251 mmol/kgH(2)O). Plasma osmolality is higher (337 +/- 5 vs. 319 +/- 3 mmol/kgH(2)O) in cPLA(2)(-/-) mice that lose a greater percentage of their body weight (20 +/- 2 vs. 13 +/- 1%) compared with cPLA(2)(+/+) mice after water deprivation. Vasopressin does not correct the concentrating defect. There is progressive reduction in urinary osmolality with age in cPLA(2)(-/-) mice. Membrane-associated aquaporin-1 (AQP1) expression, identified by immunocytochemical techniques, is reduced markedly in proximal tubules of older cPLA(2)(-/-) animals but is normal in thin descending limbs. However, Western blot analysis of kidney cortical samples revealed an equivalent AQP1 signal intensity in cPLA(2)(+/+) and cPLA(2)(-/-) animals. Young cPLA(2)(-/-) mice have normal proximal tubule AQP1 staining. Collecting duct AQP2, -3, and -4 were normally expressed in the cPLA(2)(-/-) mice. Thus mice lacking cPLA(2) develop an age-related defect in renal concentration that may be related to abnormal trafficking and/or folding of AQP1 in the proximal tubule, implicating cPLA(2) in these processes.