Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.     

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.     

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

 The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories. Received: 10 May 1997 / Revised: 1 August 1997     

Association between tree-ring and needle δ<Superscript>13</Superscript>C and leaf gas exchange in <Emphasis Type="Italic">Pinus halepensis</Emphasis> under semi-arid conditions

Associations between δ¹³C values and leaf gas exchanges and tree-ring or needle growth, used in ecophysiological compositions, can be complex depending on the relative timing of CO₂ uptake and subsequent redistribution and allocation of carbon to needle and stem components. For palaeoenvironmental and dendroecological studies it is often interpreted in terms of a simple model of δ¹³C fractionation in C₃ plants. However, in spite of potential complicating factors, few studies have actually examined these relationships in mature trees over inter- and intra-annual time-scales. Here, we present results from a 4 years study that investigated the links between variations in leaf gas-exchange properties, growth, and dated δ¹³C values along the needles and across tree rings of Aleppo pine trees growing in a semi-arid region under natural conditions or with supplemental summer irrigation. Sub-sections of tissue across annual rings and along needles, for which time of formation was resolved from growth rate analyses, showed rapid growth and δ¹³C responses to changing environmental conditions. Seasonal cycles of growth and δ¹³C (up to ~4‰) significantly correlated (P<0.01) with photosynthetically active radiation, vapour pressure deficit, air temperature, and soil water content. The irrigation significantly increased leaf net assimilation, stomatal conductance and needle and tree-ring growth rate, and markedly decreased needle and tree-ring δ¹³C values and its sensitivity to environmental parameters. The δ¹³C estimates derived from gas-exchange parameters, and weighted by assimilation, compared closely with seasonal and inter-annual δ¹³C values of needle- and tree-ring tissue. Higher stomatal conductances of the irrigated trees (0.22 vs. 0.08 mol m⁻² s⁻¹ on average) corresponded with ~2.0‰ lower average δ¹³C values, both measured and derived. Derived and measured δ¹³C values also indicated that needle growth, which occurs throughout the stressful summer was supported by carbon from concurrent, low rate assimilation. For Aleppo pine under semi-arid and irrigated conditions, the δ¹³C of tree-ring and needle material proved, in general, to be a reasonable indicator of integrated leaf gas-exchange properties.     

Visualization Analysis of CRISPR Gene-editing Knowledge Map based on Citespace

Biology Bulletin - CRISPR is an adaptive immune defense system found in bacteria and archaea that is resistant to heterologous invasive genetic material. Later studies showed that the CRISPR system...     

Reconstitution of clathrin-coated pit budding from plasma membranes

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.     

Different HCV Genotype Distributions of HIV-Infected Individuals in Henan and Guangxi,China

Background

Due to shared transmission routes, hepatitis C virus (HCV) infection is highly prevalent among people infected with human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) is associated with hepatotoxicity, leading to the negative effects on patients with HIV/HCV co-infection. In order to provide valuable information for HCV management in this particular population, we investigated the HCV genotypes in HIV-infected individuals from Henan and Guangxi, the two provinces with the most HIV-infected cases in China.

Methods

Individuals, who acquired HIV infection through various risk routes, were recruited from Henan and Guangxi. Test of antibodies against HCV (anti-HCV) was conducted, and detection of HCV RNA was performed by PCR amplification. HCV subtypes were determined by direct sequencing of amplicons, followed by phylogenetic analysis.

Results

We recruited a total of 1,112 HIV-infected people in this present study. Anti-HCV was detected from 218 (50.1%) patients from Henan and 81 (12.0%) patients from Guangxi, respectively. The highest prevalence of HIV/HCV co-infection was observed from FBDs (former blood donors) (87.2%) in Henan and IDUs (intravenous drug users) (81.8%) in Guangxi, respectively. The seroprevalence rate of HCV among people with sexual contact was significantly higher in Henan than in Guangxi (18.7% vs. 3.5%, P<0.05). The positive rate of HCV RNA in Henan and Guangxi was 30.6% (133/435) and 11.2% (76/677), respectively. Moreover, we found that 20 anti-HCV negative samples were HCV positive by PCR amplification. HCV subtype 1b (52.7%) was predominant in Henan, followed by subtype 2a (41.9%). The most frequently detected subtypes in Guangxi were 6a (35.6%) and 3b (32.9%).

Conclusion

The HCV genotype distributions were different in HIV-infected people from Henan and Guangxi. HIV/HCV co-infection was not only linked to the transmission routes, but also associated with the geographic position.     

A fine physical map of the rice chromosome 5

A fine physical map of the rice (Oryza sativa spp. Japonica var. Nipponbare) chromosome 5 with bacterial artificial chromosome (BAC) and PI-derived artificial chromosome (PAC) clones was constructed through integration of 280 sequenced BAC/PAC clones and 232 sequence tagged site/expressed sequence tag markers with the use of fingerprinted contig data of the Nipponbare genome. This map consists of five contigs covering 99% of the estimated chromosome size (30.08 Mb). The four physical gaps were estimated at 30 and 20 kb for gaps 1–3 and gap 4, respectively. We have submitted 42.2-Mb sequences with 29.8 Mb of nonoverlapping sequences to public databases. BAC clones corresponding to telomere and centromere regions were confirmed by BAC-fluorescence in situ hybridization (FISH) on a pachytene chromosome. The genetically centromeric region at 54.6 cM was covered by a minimum tiling path spanning 2.1 Mb with no physical gaps. The precise position of the centromere was revealed by using three overlapping BAC/PACs for ~150 kb. In addition, FISH results revealed uneven chromatin condensation around the centromeric region at the pachytene stage. This map is of use for positional cloning and further characterization of the rice functional genomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Chia-Hsiung Cheng and Mei-Chu Chung have equal contributions.