Divergent regulation of 1,25-dihydroxyvitamin D3 on human bone marrow osteoclastogenesis and myelopoiesis

The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has influence over osteoclastogenesis and myelopoiesis, but the regulational mechanism is not well-defined. In this report, formation of osteoclast-like (OCL) cells from primitive myeloid colony-forming cells (PM-CFC) as mediated by 1,25(OH)2D3 was examined. Our results present in this report clearly show that 1,25(OH)2D3 dose-dependently stimulated OCL cell formation when added to suspension cultures of individually replated PM-CFC colonies. Marrow cells were plated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or the human bladder carcinoma cell line 5637 conditioned medium (5637 CM) as the source of colony-stimulating activity. The 1,25(OH)2D3 effect of osteoclast differentiation was associated with a concomitant decrease in clonogenic growth of myelopoietic progenitors in response to colony-stimulating activity. Secondly, the effect of adding the known stimulator of hematopoiesis, interleukin-1beta (IL-1beta) and/or 1,25(OH)2D3 on human myeloid colony growth was assessed. IL-1beta enhanced the formation of primitive myeloid colonies in response to GM-CSF by 160%. On the other hand, 1,25(OH)2D3 dose-dependently inhibited both GM-CSF- and 5637 CM-driven myeloid colony formation by as much as 90% at 100 nM. Addition of IL-1beta to GM-CSF-stimulated cultures dampened the inhibitory effect of 1,25(OH)2D3. The inhibition of myeloid clonogenic growth by 1,25(OH)2D3 was almost abolished (89%) by simultaneously adding anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha MoAb) to the culture medium. These results collectively suggest divergent roles for 1,25(OH)2D3 in osteoclastogenesis and myelopoiesis, promoting the differentiation of OCL cells from primitive myeloid cells but inhibiting the proliferation of later myeloid progenitor cells. This inhibition of myeloid progenitors may be mediated by TNF-alpha.     

Cyclic expression of HLA class I and II molecules on the surface of purified human spermatozoa and their control by serum inhibin B levels

HLA class I and class II expression was analyzed weekly by cytofluorometry on spermatozoa samples from four donors during a 15-wk trial. On the same day that semen samples were studied, and to analyze whether this expression was hormone-controlled, serum levels of testosterone, LH, FSH, inhibin B, activin, and pro-alphaC on the one hand, and seminal plasma levels of inhibin B, activin, and alpha-inhibin on the other, were also measured. Inhibin B and related peptides were quantitated using a novel two-site assay with monoclonal antibodies to the alpha and beta subunits of inhibin. Our results showed that HLA class I and class II molecules were expressed on the spermatozoa's surface, following a cyclic pattern, and that there was a simultaneous and coordinated expression of both types of molecules (r = 0.801, P < 0.0001). Furthermore, when the expression of these molecules was plotted against the different hormone levels, serum inhibin B showed a clear inverse correlation with HLA class I (r = -0.612, P < 0.0001) and class II (r = -0.534, P < 0.0001). This finding reveals unexpected functions of inhibin B, which may be relevant in the fertilization process and on male fertility control.     

Lack of an absolute requirement for the native aryl hydrocarbon receptor (AhR) and AhR nuclear translocator transactivation domains in protein kinase C-mediated modulation of the AhR pathway.

Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CHOK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four dioxin-responsive enhancer elements. Co treatment of L10.I cells with 10 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and 81 nM phorbol 12-myristate 13-acetate (PMA), an activator of sn-1,2-diacylglyerol binding PKCs, enhanced transactivation of the reporter construct several-fold relative to cells treated with a saturating 10 nM TCDD dose alone; this effect was dubbed the "PMA effect." A domain swapping and deletional analysis of the native AhR and AhR nuclear translocator (ARNT) protein transactivation domains (TADs) was performed to determine if these domains are absolutely required for the AhR x ARNT dimer-mediated PMA effect in the L10.I model system; controls demonstrate the suitability of the L10.I model for these analyses and that endogenous AhR and ARNT levels are extremely low in this model. Transient coexpression of the AhR and ARNT-474-FLAG, an ARNT protein lacking the native ARNT TAD, in L10.I cells reveals the native ARNT TAD is not absolutely required for the AhR x ARNT-474-FLAG dimer to mediate the PMA effect. Transient coexpression of AhRDeltaCVP, a chimeric AhR protein in which the native AhR TAD has been replaced with the VP16 (herpes simplex virus protein 16) TAD (which control experiments demonstrate is unaffected by PMA), and ARNT in L10.I cells indicates that the native AhR TAD is not absolutely required for this AhRDeltaCVP x ARNT dimer to mediate the PMA effect. These observations strongly suggest that PKC-mediated modulation of the AhR pathway is not absolutely dependent on coactivators recruited to the AhR. ARNT dimer by the native TADs of the AhR and its heterodimerization partner ARNT.     

Autosomal, mitochondrial, and Y chromosome DNA variation in Finland: evidence for a male-specific bottleneck

The high prevalence of rare genetic diseases in Finland has been attributed to a founder effect some 2,000 years ago. However, this hypothesis has not been supported from mtDNA sequence and autosomal microsatellite data which indicate high levels of gene diversity. Here we have identified genetic evidence for a population bottleneck by examining variable microsatellite loci on the nonrecombining portion of Y chromosomes from Finland and four populations from Europe and the Americas. Sequence data from segment I of the control region (HVS-1) of mtDNA (360 bases) and 20 autosomal dinucleotide repeat markers were also analyzed. Partitions of genetic variance within and between populations revealed significant levels of Y-chromosome differentiation between populations. Phylogenetic and diversity analyses revealed divergent Finnish Y-haplotype clades and significantly lower Y-haplotype diversity among Finns as compared to other populations. Surprisingly, Finnish Y-haplotype diversity was even lower than the Native American populations. These results provide support for the Finnish bottleneck hypothesis. Evidence for two separate founding Finnish Y-chromosome lineages was also observed from the Y-chromosome phylogeny. A limited number of closely related founding males may have contributed to the low number of paternal lineages in the Finnish population. In contrast, high levels of genetic diversity for mtDNA and autosomal STRs may be the result of sex-biased gene flow and recent immigration to urban areas from established internal isolates within Finland.     

Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17alpha-hydroxylase-C17,20-lyase inhibitors

We have designed and synthesized a number of cytochrome P450 17alpha-hydroxylase-C17,20-lyase (P450c17) inhibitors with the aim of inhibiting androgen synthesis. To select the most potent inhibitors, we initially used human testicular microsomes, which have a high level of expression of this enzyme. However, due to lack of availability of human tissue and variability among the samples, we utilized recombinant human enzyme expressed in Escherichia coli. We designed a simple and economical protocol based on the report that recombinant bovine P450c17 can be functionally active in live bacteria. In the assay we report here, we substituted high-performance liquid chromatography product isolation with a rapid biochemical acetic acid releasing assay and utilized intact P450c17-expressing E. coli for the source of the enzyme. Enzymatic parameters of the bacterial system (Km = 5.1 x 10(-7) M, Vmax = 15.0 pmol/min/mg) were similar to those of human testicular microsomes (Km = 4.8 x 10(-7) M, Vmax = 40.0 pmol/min/mg), and our compounds displayed a similar pattern of inhibition in both systems. This new system is a fast, reliable, and reproducible method for screening P450c17 inhibitors. Furthermore, it eliminates our dependence on human tissue and potential data fluctuations caused by variations in enzymatic activity between donors.     

蛋白激酶A介导慢性压迫背根节神经元的肾上腺素能兴奋反应

本实验在奶节（ＤＲＧ）慢性压迫模型上,采用离体灌流ＤＲＧ和单纤维记录神经元自发放电的方法研究了初级感觉神经元的交感－感觉耦联作用及其细胞内机制。用外源性支甲肾上腺素（ＮＥ,１０μｍｏｌ／Ｌ）浸浴损伤的ＤＲＧ时,在９５个ＤＲＧ神经元中有８５个自发放电的神经元产生明显反应。其中,４４个呈现单纯兴奋效应,２１个表现先兴奋后抑制效应,６个出现兴奋－抑制交替振荡现象,１４个表现抑制效应。ＮＥ对损伤神经元的兴     

受损背根节神经元自发放电节律的整数倍模式及其动力学变化

本文记录了大鼠损伤背根节神经元的自发放电活动。采用背根节慢性压迫动物模型,记录慢性压迫手术后３－１０ｄ背根节的自发放电。在记录的１５６根纤维中,观察到１７根（占１１Ａ％）出现的动作电位峰峰间期以某一基础间期的整数倍模式出现的整数倍时间节律形式,其回归映射图为晶格状点阵结构,并且该时间形式受细胞膜上钠,钾通道的调控。     

FoxA proteins regulate H19 endoderm enhancer E1 and exhibit developmental changes in enhancer binding in vivo

Multiple enhancers govern developmental and tissue-specific expression of the H19-Igf2 locus, but factors that bind these elements have not been identified. Using chromatin immunoprecipitation, we have found two FoxA binding sites in the H19 E1 enhancer. Mutating these sites diminishes E1 activity in hepatoma cells. Additional chromatin immunoprecipitations show that FoxA binds to E1 in fetal liver, where H19 is abundantly expressed, but that binding decreases in adult liver, where H19 is no longer transcribed, even though FoxA proteins are present at both times. FoxA proteins are induced when F9 embryonal carcinoma cells differentiate into visceral endoderm (VE) and parietal endoderm (PE). We show that FoxA binds E1 in VE cells, where H19 is expressed, but not in PE cells, where H19 is silent. This correlation between FoxA binding and H19 expression indicates a role for FoxA in regulating H19, including developmental activation in the yolk sac and liver and postnatal repression in the liver. This is the first demonstration of a tissue-specific factor involved in developmental control of H19 expression. These data also indicate that the presence of FoxA proteins is not sufficient for binding but that additional mechanisms must govern the accessibility of FoxA proteins to their cognate binding sites within the H19 E1 enhancer.     

Cloning and tissue distribution of the human B3GALT7 gene, a member of the β1,3-Glycosyltransferase family

We report here the cloning and tissue distribution of the human B3GALT7 gene, a member of the beta1,3-Glycosyltransferase family, structurally related to the beta1,3-Galactosyltransferase family and beta1,3- N -acetylglucosaminyltransferase family, isolated from a human lung cDNA library. B3GALT7 is mapped to chromosome 19q13.2 by browsing the UCSC genomic database. It contains an ORF with length of 1191bp, encoding a protein with a signal peptide sequence and galactosyl-T domain, and its molecular weight and isoelectric point is predicted to be 43.3 kDa and 8.67 respectively. The molecular weight of the protein when expressed in E. coli corresponded to that expected. Northern blotting showed that B3GALT7 was highly expressed in lung, throat and ileum, whereas the expression level was low in tongue, breast, uteri, testis. In addition, it was also demonstrated that B3GALT7 is differentially transcribed in human tumor cell lines.