Development of polyol-responsive antibody mimetics for single-step protein purification.

The purification of functional proteins is a critical pre-requisite for many experimental assays. Immunoaffinity chromatography, one of the fastest and most efficient purification procedures available, is often limited by elution conditions that disrupt structure and destroy enzymatic activity. To address this limitation, we developed polyol-responsive antibody mimetics, termed nanoCLAMPs, based on a 16 kDa carbohydrate binding module domain from Clostridium perfringens hyaluronidase. nanoCLAMPs bind targets with nanomolar affinity and high selectivity yet release their targets when exposed to a neutral polyol-containing buffer, a composition others have shown to preserve quaternary structure and enzymatic activity. We screened a phage display library for nanoCLAMPs recognizing several target proteins, produced affinity resins with the resulting nanoCLAMPs, and successfully purified functional target proteins by single-step affinity chromatography and polyol elution. To our knowledge, nanoCLAMPs constitute the first antibody mimetics demonstrated to be polyol-responsive.

Distinct transcriptional mechanisms direct expression of the rat Dmrt1 promoter in sertoli cells and germ cells of transgenic mice.

DMRT1 is a transcription factor expressed only in Sertoli cells and undifferentiated spermatogonia of the postnatal testis, where it is required for proper cellular differentiation and fertility. To elucidate the transcriptional regulatory regions that provide DMRT1's cell-specific expression, transgenic mice containing a LacZ reporter gene driven by variable amounts of rat Dmrt1 5' flanking sequence, 9 kb and smaller, were evaluated. Examination of transgene expression by RT-PCR indicated that multiple promoter regions direct Dmrt1 to the testis and that sequences upstream of 2.8 kb are needed for both Sertoli cell expression and limiting transcriptional influence imposed by surrounding chromatin. Thus, whereas many of the transgenes were expressed in the testis, the ones with smaller promoters were significantly more prone to expression at ectopic sites or to complete silencing. Transgene expression in Sertoli cells and germ cells was assessed by immunohistochemistry and RT-PCR following busulfan treatment to remove germ cells. Both evaluations indicated expression of the 9- and 3.2-kb promoters in Sertoli cells and germ cells, whereas activity of smaller promoters was largely restricted to germ cells. In all, the present study provides in vivo evidence that distinct promoter sequences participate in Dmrt1 regulation in somatic cells and germ cells, with the -3.2 kb/-2.8 kb region directing expression in Sertoli cells and downstream sequences (< or =1.3 kb) directing it in germ cells. Further exploration of the mechanisms restricting Dmrt1 expression to the testis revealed that FOXL2, a transcription factor required for differentiation of the ovary, repressed Dmrt1 promoter through the -3.2 kb/-2.8 kb regulatory region, offering a potential mechanism for Dmrt1 transcriptional silencing in granulosa cells.

In vivo regulation of follicle-stimulating hormone receptor by the transcription factors upstream stimulatory factor 1 and upstream stimulatory factor 2 is cell specific.

Pituitary FSH promotes pubertal timing and normal gametogenesis by binding its receptor (FSHR) located on Sertoli and granulosa cells of the testis and ovary, respectively. Studies on Fshr transcription provide substantial evidence that upstream stimulatory factor (USF) 1 and USF2, basic helix-loop-helix leucine zipper proteins, regulate Fshr through an E-box within its promoter. However, despite the strong in vitro support for USF1 and USF2 in Fshr regulation, there is currently no in vivo corroborating evidence. In the present study, chromatin immunoprecipitation demonstrated specific binding of USF1 and USF2 to the Fshr promoter in both Sertoli and granulosa cells, in vivo. Control cells lacking Fshr expression showed no USF-Fshr promoter binding, thus correlating USF-promoter binding to gene activity. Evaluation of Fshr expression in Usf1 and Usf2 null mice further explored USF's role in Fshr transcription. Loss of either gene significantly reduced ovarian Fshr levels, whereas testis levels were unaltered. Chromatin immunoprecipitation analysis of USF-Fshr promoter binding in Usf-null mice indicated differences in the composition of promoter-bound USF dimers in granulosa and Sertoli cells. Promoter-bound USF dimer levels declined in granulosa cells from both null mice, despite increased USF2 levels in Usf1-null ovaries. However, compensatory increases in promoter-bound USF homodimers were evident in Usf-null Sertoli cells. In summary, this study provides the first in vivo evidence that USF1 and USF2 bind the Fshr promoter and revealed differences between Sertoli and granulosa cells in compensatory responses to USF loss and the USF dimeric composition required for Fshr transcription.

Sex-specific differences in mouse DMRT1 expression are both cell type- and stage-dependent during gonad development.

Immunohistochemistry was used to examine GCNA1, a germ cell-specific protein, together with DMRT1 (Doublesex and Mab-3-related transcription factor-1), a transcription factor implicated in Sertoli cell and germ cell function, in order to resolve DMRT1's cellular profile during pre- and postnatal gonad development in the mouse. In the indifferent gonad (10.5-11.5 days postcoitus [dpc]), DMRT1 localized to somatic cells and GCNA1(+) germ cells and was indistinguishable in males and females. By 12.5 dpc, a clear sexual preference for DMRT1 in male somatic cells was observed, with male DMRT1 localized to testicular cords and more abundant in Sertoli cells than in germ cells and female DMRT1 diffusely labeled and markedly lower in somatic cells than in germ cells. A male somatic preference continued throughout development, with DMRT1 evident in Sertoli cells at all ages examined and absent in ovarian somatic cells from 13.5 dpc onward. In contrast, expression in primordial germ cells was not sexually distinct, and both sexes showed DMRT1 increasing through 13.5 dpc and absent by 15.5 dpc. Notably, sexual differences in germ cell DMRT1 were detected after birth, when it was detected only in spermatogonia of the testis. Colocalization of DMRT1 with proliferation markers KI67 and proliferating cell nuclear antigen (PCNA) and stem cell markers OCT4 (also known as POU5F1) and NGN3 indicated that, in postnatal testes, DMRT1 was present in both stem and proliferating spermatogonia. Together, the findings implicate opposite functions for DMRT1 in somatic and germ cells of the testis. In Sertoli cells, DMRT1 expression correlated with differentiation, whereas in germ cells, it suggested a role in expansion and maintenance of undifferentiated spermatogonia.

Expression of steroidogenic factor 1 in the testis requires an interactive array of elements within its proximal promoter.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that is important for expression of genes involved in sexual differentiation, testicular and adrenal development, and hormone synthesis and regulation. To better understand the mechanisms required for SF-1 production, we employed transient transfection analysis and electrophoretic mobility shift assays to characterize the elements and proteins required for transcriptional activity of the SF-1 proximal promoter in testicular Sertoli and Leydig cells and adrenocortical cells. Direct comparison of SF-1-promoter activity in testis and adrenal cell types established that a similar set of regulatory elements (an E box, CCAAT box, and Sp1-binding sites) is required for proximal promoter activity in these cells. Further evaluation of the E box and CCAAT box revealed a novel synergism between the two elements and identified functionally important bases within the elements. Importantly, DNA/protein-binding studies uncovered new proteins interacting with the E box and CCAAT box. Thus, in addition to the previously identified USF and NF-Y proteins, newly described complexes, having migration properties that differed between Sertoli and Leydig cells, were observed bound to the E box and CCAAT box. Transient transfection analysis also identified several Sp1/Sp3-binding elements important for expression of SF-1 in the testis, one of which was previously described for expression in the adrenal gland whereas the other two were newly disclosed elements.

VLA-2 (alpha2beta1) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment.

In an attempt to identify the rotavirus receptor, we tested 46 cell lines of different species and tissue origins for susceptibility to infection by three N-acetyl-neuraminic (sialic) acid (SA)-dependent and five SA-independent rotavirus strains. Susceptibility to SA-dependent or SA-independent rotavirus infection varied depending on the cell line tested and the multiplicity of infection (MOI) used. Cells of renal or intestinal origin and transformed cell lines derived from breast, stomach, bone, or lung were all susceptible to rotavirus infection, indicating a wider host tissue range than previously appreciated. Chinese hamster ovary (CHO), baby hamster kidney (BHK-21), guinea pig colon (GPC-16), rat small intestine (Rie1), and mouse duodenum (MODE-K) cells were found to support only limited rotavirus replication even at MOIs of 100 or 500, but delivery of rotavirus particles into the cytoplasm by lipofection resulted in efficient rotavirus replication. The rotavirus cell attachment protein, the outer capsid spike protein VP4, contains the sequence GDE(A) recognized by the VLA-2 (alpha2beta1) integrin, and to test if VLA-2 is involved in rotavirus attachment and entry, we measured infection in CHO cells that lack VLA-2 and CHO cells transfected with the human alpha2 subunit (CHOalpha2) or with both the human alpha2 and beta1 subunits (CHOalpha2beta1) of VLA-2. Infection by SA-dependent or SA-independent rotavirus strains was 2- to 10-fold more productive in VLA-2-expressing CHO cells than in parental CHO cells, and the increased susceptibility to infection was blocked with anti-VLA-2 antibody. However, the levels of binding of rotavirus to CHO, CHOalpha2, and CHOalpha2beta1 cells were equivalent and were not increased over binding to susceptible monkey kidney (MA104) cells or human colonic adenocarcinoma (Caco-2, HT-29, and T-84) cells, and binding was not blocked by antibody to the human alpha2 subunit. Although the VLA-2 integrin promotes rotavirus infection in CHO cells, it is clear that the VLA-2 integrin alone is not responsible for rotavirus cell attachment and entry. Therefore, VLA-2 is not involved in the initial attachment of rotavirus to cells but may play a role at a postattachment level.