HIV-1-induced migration of monocyte-derived dendritic cells is associated with differential activation of MAPK pathways

From the site of transmission at mucosal surfaces, HIV is thought to be transported by DCs to lymphoid tissues. To initiate migration, HIV needs to activate DCs. This activation, reflected by intra- and extracellular changes in cell phenotype, is investigated in the present study. In two-thirds of the donors, R5- and X4-tropic HIV-1 strains induced partial up-regulation of DC activation markers such as CD83 and CD86. In addition, CCR7 expression was increased. HIV-1 initiated a transient phosphorylation of p44/p42 ERK1/2 in iDCs, whereas p38 MAPK was activated in both iDCs and mDCs. Up-regulation of CD83 and CD86 on DCs was blocked when cells were incubated with specific p38 MAPK inhibitors before HIV-1-addition. CCR7 expression induced by HIV-1 was sufficient to initiate migration of DCs in the presence of secondary lymphoid tissue chemokine (CCL21) and MIP-3beta (CCL19). Preincubation of DCs with a p38 MAPK inhibitor blocked CCR7-dependent DC migration. Migrating DCs were able to induce infection of autologous unstimulated PBLs in the Transwell system. These data indicate that HIV-1 triggers a cell-specific signaling machinery, thereby manipulating DCs to migrate along a chemokine gradient, which results in productive infection of nonstimulated CD4(+) cells.     

Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements

Cancer derived microarray data sets are routinely produced by various platforms that are either commercially available or manufactured by academic groups. The fundamental difference in their probe selection strategies holds the promise that identical observations produced by more than one platform prove to be more robust when validated by biology. However, cross-platform comparison requires matching corresponding probe sets. We are introducing here sequence-based matching of probes instead of gene identifier-based matching. We analyzed breast cancer cell line derived RNA aliquots using Agilent cDNA and Affymetrix oligonucleotide microarray platforms to assess the advantage of this method. We show, that at different levels of the analysis, including gene expression ratios and difference calls, cross-platform consistency is significantly improved by sequence- based matching. We also present evidence that sequence-based probe matching produces more consistent results when comparing similar biological data sets obtained by different microarray platforms. This strategy allowed a more efficient transfer of classification of breast cancer samples between data sets produced by cDNA microarray and Affymetrix gene-chip platforms.     

Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks

Loss or addition of nucleotides at junctions generated by V(D)J recombination significantly expands the antigen-receptor repertoire. Addition of nontemplated (N) nucleotides is carried out by terminal deoxynucleotidyl transferase (TdT), whose only known physiological role is to create diversity at V(D)J junctions during lymphocyte development. Although purified TdT can act at free DNA ends, its ability to add nucleotides (i.e. form N regions) at coding joints appears to depend on the nonhomologous end-joining factor Ku80. Because the DNA ends generated during V(D)J rearrangements remain associated with the RAG proteins after cleavage, TdT might be targeted for N region addition through interactions with RAG proteins or with Ku80 during remodeling of the post-cleavage complex. Such regulated access would help to prevent TdT from acting at other types of broken ends and degrading the fidelity of end joining. To test this hypothesis, we measured TdT’s ability to add nucleotides to endonuclease-induced chromosomal and extrachromosomal breaks. In both cases TdT added nucleotides efficiently to the cleaved DNA ends. Strikingly, the frequency of N regions at non-V(D)J-generated ends was not dependent on Ku80. Thus our results suggest that Ku80 is required to allow TdT access to RAG post-cleavage complexes, providing support for the hypothesis that Ku is involved in disassembling or remodeling the post-cleavage complex. We also found that N regions were abnormally long in the absence of Ku80, indicating that Ku80 may regulate TdT’s activity at DNA ends in vivo.     

Laser-assisted microdissection of the kidney: Fundamentals and applications

Laser-assisted microdissection (LAM) permits the procurement of relatively pure cell populations from histological sections. When applied to the kidney, LAM combined with molecular biological techniques has expanded our understanding of renal biology and pathology. Both frozen and fixed renal tissues can be microdissected. However, sample type and tissue processing can influence the quality of molecular data generated. Data analysis may also be complicated by relative variations in gene expression levels. Importantly, preliminary studies have shown that molecular data obtained following LAM on the kidney can offer new diagnostic and prognostic information. Thus, LAM and molecular markers may eventually become incorporated into the routine kidney biopsy examination.     

Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron

The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.     

Network of protein interactions within the Drosophila inner kinetochore

The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function.     

Effects of Desiccation on Phosphorus and Potassium Acquisition by a Desiccation-tolerant Moss and Lichen

The hypothesis that desiccation-tolerant mosses and lichensmay be more responsive to nutrient inputs accompanying intermittentdesiccation than mesophytic forest species was investigatedemploying species from semi-arid grassland in Hungary. Shootapices of the moss Syntrichia ruralis and marginal lobes ofthe lichen Cladonia convoluta were maintained for 7 weeks undercontrolled conditions. They were cultivated with or withouta weekly application of the major inorganic macronutrients,and either under constant hydration or with one or two 24 hperiods of desiccation each week. Growth of S. ruralis was stimulatedby nutrient additions, but lower weight increments were achievedwith increasing frequency of desiccation. All samples of thelichen showed positive growth, and no significant treatmenteffects were detected. A large net uptake of P occurred in nutrient-treatedmaterial of both species that was unaffected by the impositionof desiccation treatments. A smaller net uptake of K into theintracellular fraction was also observed when nutrients wereapplied, but in the moss this was against a baseline of decreasingK content. In contrast, more of the original K content was retainedin C. convoluta. In neither species was any clear evidence foundfor inhibition of nutrient uptake by the desiccation episodes.It is suggested that the lack of growth response in the lichenarises from an inability to bring together the additional nutrients,presumably mainly absorbed by the mycobiont, with photosynthateproduced by the photobiont. Copyright 2000 Annals of BotanyCompany Cladonia convoluta, Syntrichia ruralis, desiccation tolerance, mineral nutrition, phosphorus, potassium     

Conservation of the Caenorhabditis elegans timing gene clk-1 from yeast to human: a gene required for ubiquinone biosynthesis with potential implications for aging

Mutations in the Caenorhabditis elegans gene clk-1 have a major effect on slowing development and increasing life span. The Saccharomyces cerevisiae homolog COQ7 encodes a mitochondrial protein involved in ubiquinone biosynthesis and, hence, is required for respiration and gluconeogenesis. In this study, RT-PCR and 5′ RACE were used to isolate both human and mouse clk-1/COQ7 homologs. Human CLK-1 was mapped to Chr 16(p12–13.1) by Radiation Hybrid (RH) and fluorescence in situ hybridization (FISH) methods. The number and location of human CLK1 introns were determined, and the location of introns II and IV are the same as in C. elegans. Northern blot analysis showed that three different isoforms of CLK-1 mRNA are present in several tissues and that the isoforms differ in the amount of expression. The functional equivalence of human CLK-1 to the yeast COQ7 homolog was tested by introducing either a single or multicopy plasmid containing human CLK-1 cDNA into yeast coq7 deletion strains and assaying for growth on a nonfermentable carbon source. The human CLK-1 gene was able to functionally complement yeast coq7 deletion mutants. The protein similarities and the conservation of function of the CLK-1/clk-1/COQ7 gene products suggest a potential link between the production of ubiquinone and aging. Received: 15 March 1999 / Accepted 11 June 1999