Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes

Unfixed metaphase chromosome preparations from human lymphocyte cultures were immunofluorescently labelled using antibodies to defined histone epitopes. Both mouse monoclonal antibody HBC-7, raised against the N-terminal region of H2B, and rabbit serum R5/12, which recognizes H4 acetylated at Lys-12, gave non-uniform labelling patterns, whereas control antibodies against total histone fractions H4 and H1 produced homogeneous fluorescence. HBC-7 bound approximately uniformly to the bulk of the chromosomes, but the major heterochromatic domains of chromosomes 1, 9, 15, 16 and the Y showed significantly brighter fluorescence. Serum R5/12 indicated an overall reduction in acetylation of H4 in metaphase chromosomes compared with interphase nuclei, although some specific chromosomal locations had considerably elevated acetylation levels. Acetylation levels in the major heterochromatic domains appeared extremely low. To investigate further the differences noted in heterochromatin labelling, metaphases from cultures grown in the presence of various agents known to induce undercondensation of the major heterochromatic domains were similarly immunolabelled. Decondensed heterochromatin no longer exhibited higher than normal immunofluorescence levels with HBC-7. The higher resolution afforded by stretching the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains. We consider the implications of these observations in relation to chromatin conformation and activity.by W.C. Earnshaw     

Immunohistochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat reproductive tissues

Vitamin D₃, via its active metabolite 1,25-dihydroxyvitamin D₃, plays a critical part in male and female reproduction in the rat. 1,25-Dihydroxyvitamin D₃ activity is mediated by an intracellular receptor (VDR). VDR distribution in reproductive tissue has not been studied using antibodies against the receptor. We developed a polyclonal antibody against the VDR and used it to examine VDR distribution in male and female rat reproductive tissues. In rat testes, VDR epitopes were observed in seminiferous tubules, specifically in spermatogonia, Sertoli cells and spermatocytes. Spermatozoa stained faintly. Epithelial cells of the epididymis, seminal vesicles and prostate also expressed VDR epitopes. In the female rat reproductive tract, immunostaining for VDR was seen in ovarian follicles, specifically in granulosa cells. Weaker VDR immunostaining was observed in follicular thecal cells and in the ovarian stroma and germinal epithelium. Corpus luteal cells stained intensely for VDR. Epithelium of fallopian tubes and the uterus also contained VDR epitopes. Both nuclear and cytoplasmic VDR immunostaining was observed in male and female rat reproductive tissues. We conclude that the VDR is widely distributed in male and female reproductive tissues and that it is likely to mediate actions of 1,25-dihydroxyvitamin D₃ in the tissues.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)(2)D(3). I. Chick intestine

Antisera were raised against the NH(2)-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3); BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [(14)C]1, 25(OH)(2)D(3)-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)(2)D(3)-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)(2)D(3) for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by electron microscopy, whereas 24, 25-dihydroxyvitamin D(3) failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)(2)D(3) confirmed a hormone-induced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)(2)D(3) has been identified.     

Distribution of 1,25-Dihydroxyvitamin D3 Receptor Immunoreactivity in the Rat Olfactory System

1.The rat olfactory system contains numerous target sites for 1,25-dihydroxyvitamin D₃, as determined by receptor protein (VDR) immunocytochemistry and in situ hybidization.2.Nuclear and cytoplasmic VDR immunoreactivity as well as the corresponding hybridization signal was observed in neurons in the olfactory epithelium, the olfactory bulb, and throughout the limbic system in locations also known to be glucocorticoid targets.3.The widespread distribution of VDR indicates the distinct functional importance of 1,25-dihydroxyvitamin D₃ for olfactory perception.     

Antibodies Directed against Functional Sites on the Acetylcholine Receptor from Torpedo Marmorata

Abstract

Antibodies directed against functional sites on the acetylcholine receptor from Torpedo marmorata have been obtained by the following two procedures: (i) Our library of monoclonal antibodies raised against the whole receptor protein was screened for antibodies competing with cholinergic agonists, antagonists and local anesthetics for receptor binding, (ii) antibodies were raised against short peptides matching the sequence of predetermined sites on the receptor protein. In this way, a topographic map of the functional sites on the receptor surface can be constructed.     

Generation and characterization of polyclonal and monoclonal antibodies to human NTPDase2 including a blocking antibody

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) is an ectonucleotidase that modulates P2 receptor activation by hydrolyzing ATP to ADP. In rodents, NTPDase2 is expressed by several specialized cell types such as vascular adventitial cells, neuroglial cells, hepatic portal fibroblasts, gustatory type I cells, and cells within the connective tissues of reproductive and gastrointestinal organs. Much less is known regarding the expression and function of NTPDase2 in humans. Here, we developed specific research tools to study human NTPDase2. We generated mouse monoclonal antibodies and rabbit polyclonal antibodies specific to human NTPDase2 and validated their specificity by western blot, immunocytochemistry, immunohistochemistry, and flow cytometry. In addition, one monoclonal antibody named hN2-D5 _s specifically inhibits human NTPDase2 enzymatic activity but not mouse nor rat NTPDase2. Using these antibodies, NTPDase2 immunoreactivity was detected on glial cells of the human enteric nervous system suggesting a function of the enzyme in intestinal motility. In conclusion, the new antibodies described in our work are novel tools that will enhance future studies of NTPDase2 expression and function in humans.     

Immunohistochemical Analysis of 1,25-dihydroxyvitamin D3 Receptor in Cervical Carcinoma

The immunohistochemical localization and expression of 1,25-dihydroxyvitamin D₃ receptors (VDR) has been investigated in normal human cervical tissue (n = 15) and in cervical carcinomas (n = 23). VDR immunoreactivity (monoclonal antibody 9A7r354;) was compared with the staining patterns of transglutaminase K, cytokeratin 10 and Ki-67 in these tumours. Moderate to strong nuclear immunoreactivity for VDR was detected in almost all cervical carcinomas analysed. VDR staining was homogeneous, with no visual differences between individual tumour cells. Some 60% of normal cervical tissues revealed weak immunoreactivity for VDR. In normal cervical tissue, nuclear VDR staining was confined to the lower cervical layers, predominantly to the basal cell layer. Both the intensity of VDR immunostaining and the number of VDR-positive cells were up-regulated in cervical carcinomas compared with normal cervical tissue. No visual correlation wa s found for the coexpression of VDR with markers of proliferation and differentiation. Our findings indicate that: (1) cervical tissue may be a new target organ for therapeutically applied vitamin D analogues; (2) VDR is up-regulated at the protein level in cervical carcinomas compared with normal cervical tissue; (3) up-regulation of VDR in cervical carcinoma is induced not exclusively by alterations in epithelial differentiation or proliferation, but by different, unknown mechanisms; and (4) calcitriol and new vitamin D analogues exerting fewer calcaemic side-effects may be promising new drugs for the treatment or chemoprevention of metastasizing cervical carcinomas as well as of cervical precancerous lesions.     

Identification of a highly specific and versatile vitamin D receptor antibody

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is critical for regulation of serum calcium and phosphorus levels and for proper maintenance of bone mineralization and neuromuscular function. Biological effects of 1,25(OH)₂D₃ are mediated through a nuclear steroid hormone receptor, known as the vitamin D receptor (VDR). The discovery of VDR in a number of different cell and tissue types, suggests that the physiological role of vitamin D may extend beyond the regulation of calcium homeostasis and bone function. Unfortunately, identification of tissues expressing VDR has been controversial due to low abundance of the receptor and quality of the antibodies used. Therefore, we elected to characterize a panel of commercially available VDR antibodies in order to identify antibodies with high specificity and sensitivity. To address these objectives, we have used multiple immunoassays to determine VDR expression in tissues from several organs from multiple species employing tissues from VDR knockout mice as critical negative controls. Many of the antibodies tested showed nonspecific binding that can account for divergent reports. However, one antibody, identified as D-6, is highly specific and extremely sensitive. The specificity, sensitivity, and versatility of this antibody make it the preferred antibody for identifying VDR expression in target tissues using immunological methods.     

Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human α2

23 hybridomas secreting monoclonal antibodies against human ₂ , the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class tgG2b. All light chains belong to the group. The specificity and relative avidity of these monoclonals have been determined Using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for ₂ , 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 rnonoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the ₂ molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.     

The importance of nuclear import in protection of the vitamin D receptor from polyubiquitination and proteasome‐mediated degradation

Others and we previously showed that the vitamin D receptor (VDR) is subject to degradation by the 26S proteasome and that treatment with 1,25‐dihydroxyvitamin D₃ (1,25D₃) inhibited this degradation. In the present study, we found that in osteoblasts, but not in intestinal epithelial cells, the VDR was susceptible to degradation by the 26S proteasome. The subcellular site for degradation of the VDR in osteoblasts is the cytoplasm and the site for ligand‐dependent protection of the VDR from the 26S proteasome is the chromatin. These direct relationships between nuclear localization and protection of the VDR from 26S proteasome degradation led us to hypothesize that the unoccupied cytoplasmic VDR is a substrate for polyubiquitination, which targets VDR for degradation by the 26S proteasome, and that nuclear localization has the ability to protect the VDR from polyubiquitination and degradation. To test these hypotheses, we used Cos‐1 cells transfected with human VDR and histidine‐tagged ubiquitin expression vectors. We found that unoccupied VDR was polyubiquitinated and that 1,25D₃ inhibited this modification. Mutations in the nuclear localization signal of VDR (R49W/R50G and K53Q/R54G/K55E) or in the dimerization interface of VDR with retinoid X receptor (M383G/Q385A) abolished the ability of 1,25D₃ to protect the VDR from polyubiquitination, although these mutations had no effect on the ligand‐binding activity of VDR. Therefore, we concluded that in some cellular environments unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation and that ligand‐dependent heterodimerization and nuclear localization protect the VDR from these modifications. J. Cell. Biochem. 110: 926–934, 2010. © 2010 Wiley‐Liss, Inc.