It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2′-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy.

Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM.

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP180, in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP180 concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP180 is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.

To improve our understanding of the roles of microtubule cross-linking motors in mitosis, we analyzed two sea urchin embryonic kinesin-related proteins. It is striking to note that both of these proteins behave as homotetramers, but one behaves as a more compact molecule than the other. These observations suggest that these two presumptive motors could cross-link microtubules into bundles with different spacing. Both motors localize to mitotic spindles, and antibody microinjection experiments suggest that they have mitotic functions. Thus, one of these kinesin-related proteins may crosslink spindle microtubules into loose bundles that are "tightened" by the other.Animal cell reproduction involves mitosis and cytokinesis, events that depend on the action of a bipolar protein machine, the mitotic spindle, which uses microtubules (MTs) 1 and MTbased motor proteins. A classic model system for studying mitosis and cytokinesis is the early echinoderm embryo where MTs and MT-based motor proteins are thought to position mitotic centrosomes, centrosomes dictate the positioning of the spindle, and the spindle in turn positions the cleavage plane (1). Antibody microinjection experiments are useful for probing the functions of MT-motors in sea urchin embryonic cell division (2); previously, the microinjection of pan-kinesin antibodies suggested that some kinesin motors, but apparently not conventional kinesin or heterotrimeric kinesin II, play important roles in mitosis and cell division (3-6).One of these motors is a 110-kDa polypeptide, KRP 110 , that reacts with an antibody, CHO1, to the mammalian mitotic motor, MKLP1 (3). MKLP1 is an anti-parallel MT-MT sliding motor that appears to be required for the organization of midzonal MTs and progression through mitosis and cytokinesis in several systems (7-12). The mechanism of how MKLP1 family members might function to cross-link microtubules and bundle them into the anti-parallel arrays required for cytokinesis is still unclear, because the size and subunit composition of the native holoenzyme is unclear.The microinjection of the CHO1 antibody into sea urchin embryonic cells caused a prophase or metaphase arrest, suggesting that KRP 110 is a functional homologue of MKLP1 and is required for mitosis and cell division in this system (3). Work done in several systems suggests that interdigitating MTs in the spindle midzone are required to signal the proper progression of the contractile ring and completion of cytokinesis. However, in echinoderm cells, cleavage furrows can form, and subsequent cytokinesis can occur between two adjacent MT asters in the absence of such midzonal MTs (1). This raises the possibility that the MKLP1 homologue in echinoderm embryos, KRP 110 , may be localized to the MT asters rather than the central spindle (13).Another putative MT cross-linking motor that is also likely to participate in sea urchin embryonic mitosis is KRP 170 , a member of the phylogenetically diverse bipolar bimC kinesin subfamily. Bipolar bimC kinesins are homotetramers that move ...

Viral regulatory complexes perform critical functions during virus replication and are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form complexes with multiple viral and cellular factors to direct transcription and export of the viral RNA. These complexes are composed of many proteins and are dynamic, making them difficult to fully recapitulate in vitro. Therefore, we developed a cell-based reporter assay to monitor the assembly of viral complexes for inhibitor screening. We screened a small-molecule library and identified multiple hits that inhibit the activity of the viral complexes. A subsequent chemistry effort was focused on a thieno[2,3-b]pyridine scaffold, examples of which inhibited HIV replication and the emergence from viral latency. Notable aspects of the effort to determine the structure-activity relationship (SAR) include migration to the regioisomeric thieno[2,3-c]pyridine ring system and the identification of analogs with single-digit nanomolar activity in both reporter and HIV infectivity assays, an improvement of Ͼ100-fold in potency over the original hits. These results validate the screening strategy employed and reveal a promising lead series for the development of a new class of HIV therapeutics.KEYWORDS antivirals, HIV, RNA, Rev R NA-protein complexes are essential to the assembly and activity of many viral regulatory systems and represent an important target class for antiviral drug discovery. In HIV, the Rev-Rev response element (RRE) protein-RNA complex is one such target due to its essential activity in mediating the export of unspliced and partially spliced RNAs from the nucleus to the cytoplasm (1, 2). Disrupting the Rev-RRE interaction prevents the expression of late viral proteins and the packaging of viral RNA, thus inhibiting virus replication.Rev is a 116-amino-acid RNA-binding protein that is expressed from fully spliced mRNAs early in the virus life cycle (3). Rev binds the RRE, a highly structured ϳ350-nucleotide (nt) RNA element encoded within the env gene (reviewed in references 2 and 4). Current models suggest that about six Rev molecules bind the RNA in order to properly position two of the nuclear export sequences on Rev for binding to a dimer of the Crm1-RanGTP export complex (5, 6). This complex is then exported through the nuclear pore, after which it disassembles in the cytoplasm to allow translation of the late HIV proteins and packaging of the viral genome (2, 7).The formation of the export complex is driven by several critical intermolecular interactions. Rev binds the RNA primarily through its arginine-rich motif (ARM), an ␣-helical domain that forms several important hydrogen bonds with the RNA (8, 9). The nuclear magnetic resonance (NMR) structures of the Rev peptide complexed to RRE IIB or to an RNA aptamer and a recent cocrystal structure of a Rev-RRE dimer (10) show that