Zhijian Lu

CXCR-4 Desensitization Is Associated with Tissue Localization of Hemopoietic Progenitor Cells

The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34+ cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34+ cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1β analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin−, Sca-1+, Thy-1low, and c-kit+ hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation.

Molecular Analysis of a Novel Winged Helix Protein, WIN EXPRESSION PATTERN, DNA BINDING PROPERTY, AND ALTERNATIVE SPLICING WITHIN THE DNA BINDING DOMAIN

We have cloned a novel winged helix factor, WIN, from the rat insulinoma cell line, INS-1. Northern blot analysis demonstrated that WIN is highly expressed in a variety of insulinoma cell lines and rat embryonic pancreas and liver. In adults, WIN expression was detected in thymus, testis, lung, and several intestinal regions. We determined the DNA sequences bound in vitro by baculovirus-expressed WIN protein in a polymerase chain reaction-based selection procedure. WIN was found to bind with high affinity to the selected sequence 5′-AGATTGAGTA-3′, which is similar to the recently identified HNF-6 binding sequence 5′-DHWATTGAYTWWD-3′ (where W = A or T, Y = T or C, H is not G, and D is not C). We have isolated human WIN cDNAs by library screening and 5′-rapid amplification of cDNA ends. Sequence analysis indicates that the carboxyl terminus of human WIN has been previously isolated as a putative phosphorylation substrate, MPM2-reactivephosphoprotein 2 (MPP2); WIN may be regulated by phosphorylation. Alignment of the rat and human WIN cDNAs and their comparison with mouse genomic sequence revealed that the WIN DNA binding domain is encoded by four exons, two of which (exons 4 and 6) are alternatively spliced to generate at least three classes of mRNA transcripts. These transcripts were shown by RNase protection assay to be differentially expressed in different tissues. Alternative splicing within the winged helix DNA binding domain might result in modulation of DNA binding specificity.

[21] Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli

Publisher Summary In this chapter, protocols are provided for the use of thioredoxin gene fusion expression system. It describes a variety of suitable E. coli expression strains and a number of thioredoxin expression vectors. It also presents the procedures for thioredoxin fusion protein purification and method for specific cleavage of thioredoxin fusions by enterokinase. Thioredoxin fusions have proved to be especially useful in avoiding inclusion body formation, particularly for the production of small, normally secreted, mammalian cytokines in an active form in the E. coli cytoplasm. E. coli thioredoxin is a compact, highly soluble, and thermally stable protein with robust folding characteristics. These properties perhaps allow the molecule, when fused to a protein of interest, to serve as a covalently joined molecular chaperon. Thioredoxin may, thus, act to prevent the aggregation and precipitation of fused nascent proteins, giving them an extended opportunity to adopt their correct tertiary folds. Thioredoxin also possesses a number of additional characteristics that suit it for the role as a fusion partner. It is small, highly translated, and its tertiary structure reveals that both its amino and carboxyl termini are accessible for potential fusions to other molecules. Moreover, its active site comprises a surface-accessible loop that can be utilized for internal peptide insertions. Purifications of thioredoxin fusion proteins can be facilitated by making use of the remarkable ability of the molecule to be released from the bacterial cytoplasm by simple osmotic shock, by taking advantage of the inherent thermal stability of the molecule, by using avidin or streptavidin matrices to capture thioredoxin variants modified to allow for in vivo biotinylation, or by using the engineered forms of thioredoxin with affinity to metal chelate column matrices.

Small molecules for small minds? The case for biologic pharmaceuticals.

Biologic pharmaceuticals are gaining in both market share and clinical utility compared with small molecule therapeutics. This market growth is, in part, reflective of a field of science entering its toddlerhood, where with increased maturity, both development timelines and costs of manufacturing for these complex molecules will decrease, further enhancing the profitability side of the equation. Although a firm understanding of the rules governing toxicity (especially antibody responses to therapeutic proteins) remains to be defined, it is clear that proteins are less prone to much of the idiosyncratic toxicity associated with small molecule drug candidates. Proteins are disadvantaged in that they are unlikely to find much use in targeting intercellular processes; however, they have clear strengths over small molecules in targeting protein–protein interactions and the specific targeting of surface features of particular cells (e.g., in oncology). As each aspect of protein pharmaceutical technology advances, it is clear that this will be the major area for growth in the industry over the next decade.

The CXC-chemokine, Hi74: expression in the central nervous system

H174 is a new member of the CXC-chemokine family. A cDNA probe containing the entire H174 coding region recognized a predominant inducible transcript of approximately 1.5 kb expressed in interferon (IFN) activated astrocytoma and monocytic cell lines. H174 message can be induced following IFN-alpha, IFN-beta, or IFN-gamma stimulation. H174 message was also detected in IFN treated cultures of primary human astrocytes, but was absent in unstimulated astrocytes. H174, like IP10 and Mig, lacks the ELR sequence associated with the neutrophil specificity characteristic of most CXC-chemokines. Preliminary experiments suggest H174, IP10 and Mig are independently regulated. Recombinant H174 is a weak chemoattractant for monocyte-like cells. H174 can also stimulate calcium flux responses. The data support the classification of H174 as a member of a subfamily of interferon-gamma inducible non-ELR CXC-chemokines. Brain tissues were obtained at autopsy from one patient with AIDS dementia, one patient with multiple sclerosis, and two normal control patients. H174 and Mig were detected by RT-PCR in brain tissue cDNA derived from the patients with pathological conditions associated with activated astrocytes but not in cDNA from control specimens.