Uroplakin I: a 27-kD protein associated with the asymmetric unit membrane of mammalian urothelium.

The luminal surface of mammalian urothelium is covered with numerous plaques (also known as the asymmetric unit membrane or AUM) composed of semi-crystalline, hexagonal arrays of 12-nm protein particles. Despite the presumed importance of these plaques in stabilizing the urothelial surface during bladder distention, relatively little is known about their protein composition. Using a mouse mAb, AE31, we have identified a 27-kD protein that is urothelium-specific and is differentially expressed in superficial umbrella cells. This protein (pI approximately 5.8) partitions into the detergent phase during Triton X-114 phase separation. Pulse-chase experiments using cultured bovine urothelial cells showed that this protein is synthesized as a 32-kD precursor that is processed through a 30-kD intermediate, to the mature 27-kD form. In cytoplasmic vesicles containing immature AUM, the AE31 epitope is detected in patches on the cytoplasmic side, but in mature, apical AUM it is detected exclusively on the luminal side. This suggests an unusual translocation of the AE31 epitope during AUM maturation; more data are required, however, to substantiate this interpretation. Immunoaffinity purification of the 27-kD protein results in the copurification in approximately molar ratio of a 15-kD protein, as well as a small and variable amount of a 47-kD protein. Immunoblotting data indicate that these three proteins are immunologically distinguishable. This copurified 15-kD protein is relative basic (pI approximately 8.0). Like the 27-kD protein, it is urothelium-specific and is present mainly in the umbrella cells. Together, our data indicate that a 27-kD protein is urothelial plaque-associated (uroplakin I). Based on complex formation data, we provisionally name the 15-kD protein uroplakin II; additional data will be required to determine whether this and the 47-kD protein are integral parts of AUM. The identification of these AUM-associated and -related proteins, plus the availability of a culture system capable of synthesizing and processing some of these molecules, offer new opportunities for studying the detailed structure, assembly, and function of asymmetrical unit membrane.

Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10.

The human breast epithelial cell line MCF-10 was derived from s.c. mastectomy tissue from a 36-year-old, parous, premenopausal woman with fibrocystic disease. It was initiated as a mortal cell line (MCF-10M), which senesces when transferred serially in 1.05 mM calcium. These cells spontaneously gave origin to two immortal sublines, MCF-10A, or attached cells, and MCF-10F, or floating cells, which have proliferated for more than 4 years in Dulbecco's modified essential medium and Ham's F-12 either with the customary calcium concentration of 1.05 mM (DMEM-H) or in medium containing 0.04 mM calcium or low calcium. Studies reported here indicate that MCF-10 is a mammary epithelial cell line. Electron microscopy showed that both MCF-10A and MCF-10F have characteristics of luminal ductal cells, but not of myoepithelial cells. When grown for more than 1200 days in Dulbecco's modified essential medium-Ham's F-12 and low calcium media, respectively, they maintained their epithelial characteristics, although the concentration of calcium exerted a powerful influence on cell morphology. Cells grown in Dulbecco's modified Eagle's medium-Ham's F12 medium are low cuboidal with numerous desmosomes and short microvilli, whereas those grown in low calcium medium have significantly reduced number of desmosomes, are more spherical, and have greater numbers of microvilli which are longer than those of cells grown in DMEM-H. The breast epithelial orgin of these cells was confirmed by immunocytochemical detection of epithelial sialomucins and keratins. The monoclonal antibodies MFA-breast and MC5 and the polyclonal antibody epithelial membrane antigen were used to detect the epithelial sialomucins. Keratins were characterized by using KA-4, K-14, AE1/AE3, and K-19 specific antibodies. It was concluded that MCF-10A and MCF-10F cells are breast epithelial cells and that they represent an important tool for studies of the basic processes of growth and carcinogenesis.

ABO blood type predicts the cytolocalization of anti-P-glycoprotein monoclonal antibody reactivity in human colon and ureter.

Classic multidrug resistance is mediated by a P-glycoprotein. Using monoclonal antibody C219 (MAb C219) in an immunohistochemical study, we found high levels of putative Golgi P-glycoprotein in normal columnar and transitional epithelium in subpopulations of patients with specific blood types. For example, Golgi staining was present in blood type A patients in 46% of normal colon samples (N = 21) and 88% of normal ureter samples (N = 17). In comparison, Golgi staining was present in blood group O patients in only 6% of normal colon samples (N = 34) and in 0% of normal ureter samples (N = 19). The association of MAb C219 Golgi staining with blood type A and lack of Golgi staining with blood type O was statistically significant in normal colon (P = .001) and normal ureter (P less than .0001). Inappropriate hyperexpression of P-glycoprotein was frequently found in colon carcinomas. Additional evidence that Golgi MAb C219 reactivity represents P-glycoprotein is presented. This includes (1) immunostaining of Golgi with two anti-P-glycoprotein MAbs, C219 and JSB-1, and (2) experiments in which Mab C219 Golgi reactivity was blocked by preincubation of MAb C219 with a specific P-glycoprotein epitope-containing peptide. The high degree of association of Golgi P-glycoprotein with blood type A may suggest a role for P-glycoprotein in processing or trafficking of specific blood group antigens.

Ubiquitous and cell-type-specific protein interactions with human papillomavirus type 16 and type 18 enhancers.

We have studied the protein-DNA interactions of human papillomavirus types 16 and 18 constitutive enhancer elements using DNasel footprinting experiments with nuclear extracts from four cervical carcinoma cell lines (C33A, HeLa, SiHa, and CaSki) and one fibroblast cell line (143B). Among nine footprints for the HPV 16 enhancer region, six footprints contain nuclear factor 1 (NF1) binding GCCAA motif. In vitro competition experiments suggest that the same factors are shared by all six of these motifs. Two other sequence motifs have consensus sequences for transcription factor AP1. Another sequence motif, for which uv crosslinking studies reveal interaction with four protein molecules, is a strong positive modulator of HPV 16 enhancer function in vivo and shares 100% homology to a sequence motif, GTTTTAA, in the tissue-specific enhancer of the c-mos oncogene. Footprints on the HPV 18 enhancer show five protected regions with homologies to NF1, AP1 and EFII transcription factor binding motifs. One sequence motif of the HPV 18 enhancer has three repeats of a TTTTA sequence contained within the c-mos sequence motif and interacts with at least four different individual polypeptides, as judged by uv crosslinking experiments.

Effect of fatty acid profiles on the susceptibility of cultured rabbit tracheal epithelial cells to hyperoxic injury.

To investigate the role of cellular fatty acid content on the susceptibility of airway epithelial cells to hyperoxic injury, monolayer cultures of rabbit tracheal epithelial (TE) cells were grown to confluence in serum-free media with or without a commercial mixture of cholesterol esters and phospholipid-rich lipoproteins (Excyte III, Miles-Pentex, Kankakee, IL) in conjunction with arachidonic acid complexed to BSA. Monolayer cultures were then exposed to control (5% CO2/air) or hyperoxic atmospheres (95% oxygen/5% CO2) for 2 h using an in vitro system in which cells were maintained at a gas-liquid interface analogous to in vivo conditions. Hyperoxic injury was assessed by cell viability (trypan blue exclusion) and by the generation of lipid peroxides measured as thiobarbituric acid (TBA) reactive substances. Changes in TE cell and cell culture effluent fatty acid content induced by exposure to control or hyperoxic atmospheres were analyzed by gas chromatography. TE cells grown in lipid-unsupplemented media had fatty acid profiles characteristic of essential fatty acid deficiency, whereas the fatty acid content of lipid-supplemented TE cells more closely resembled those of acutely recovered TE cells. Lipid-unsupplemented cells were more susceptible to hyperoxic injury as demonstrated by decreased viability and increased production of TBA-reactive substances compared to cells maintained in lipid-supplemented media. In both lipid-supplemented and unsupplemented cells, hyperoxic exposure was associated with a decreased relative cellular content of the monounsaturated and polyunsaturated fatty acids (PUFA) and an increased content of saturated fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogold localisation of laminin in normal and exfoliative iris.

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium.

The immunocytochemical localization of angiotensinogen in the rat ovary.

The present study examined the presence and cellular distribution of angiotensinogen, the precursor to the angiotensin peptides, in the ovary of the normal cycling rat by immunocytochemistry. Angiotensinogen staining was present in the granulosa cells of maturing follicles and to a lesser extent in those undergoing atresia. Staining was not seen in the granulosa cells of primordial or early primary follicles. In maturing follicles intense staining for angiotensinogen was confined to the antral cell layers, cells of the cumulus oophorus and in the follicular fluid. Strong immunostaining was also seen in the germinal epithelium covering the ovary. Lighter angiotensinogen staining was observed in some parts of the cortical and medullary stroma and occasionally in corpora lutea. No variation in the intensity or pattern of angiotensinogen staining was observed throughout the estrous cycle. Comparison of the distribution of angiotensinogen with the previously described localization of renin, AII, angiotensin converting enzyme and AII receptors, suggests that there are a number of intra-ovarian sites at which AII could be produced.

Effect of EDTA on cytokeratin detection in the inner ear.

Immunohistochemical studies on the epithelium of the adult inner ear are difficult to perform without decalcification of the bony capsule. In this study, we examined the effect of decalcifying agents on the immunoreactivity of various cytokeratin antigens in the cochlear duct epithelium of 2-day-old rats, allowing the comparison of fresh and decalcified specimens. Decalcification of unfixed tissue in a solution containing EDTA or EGTA and polyvinylpyrrolidone, at pH 7.4 and 4 degrees C for a maximum period of 2 days, not only preserved the antigen epitopes but even enhanced the staining intensities in comparison with fresh specimens. This enhancement effect, caused by chelating agents and found to be blocked by prior fixation with acetone, is suggested to be caused by unmasking of the antigenic epitopes.

Keratin subtypes in carcinomas of the uterine cervix: implications for histogenesis and differential diagnosis.

Normal epithelia and carcinomas of the human uterine cervix were studied by monoclonal antibodies chain specific for cytokeratins 4, 8, 10, 13, 14, 18, and 19. Most cells in 13 examined squamous carcinomas revealed a cytokeratin phenotype detected in ectocervical basal cells and endocervical subcolumnar reserve cells: 8+, 14+, 18+, 19+, 4-, 10-, 13-. We propose that these two cell types are closely related or identical and that squamous carcinoma of the cervix originates in this cell type. In more differentiated tumor cells cytokeratins 4, 10, and 13, which are present in suprabasal layers of the normal ectocervical epithelium, were coexpressed with basal cell cytokeratins. Thus, contrary to previous beliefs, all cytokeratins detected in carcinomas were also present in normal epithelium of uterine cervix. The cytokeratin profile of cervical adenocarcinomas corresponded to that of columnar endocervical cells (8+, 18+, 19+), although two of the three adenocarcinomas also expressed cytokeratin 4, which in the normal endocervix was detected in scattered single columnar cells only. The new monoclonal antibody DE-K14, specific for cytokeratin 14, proved a specific marker of subcolumnar reserve cells in the endocervix. It was also the only one that reacted with all cervical squamous carcinomas but with none of the cervical adenocarcinomas and, as such, has a potential value for pathological differential diagnosis of cervical tumors.

Characterization, in some human breast cancer cell lines, of gastrin-releasing peptide-like receptors which are absent in normal breast epithelial cells.

The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.

Polarization of gelsolin and actin binding protein in kidney epithelial cells.

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.

Nonneoplastic and nonhyperplastic thymus in myasthenia gravis. An immunohistochemical study with double immunoenzymatic labeling of basement membrane and cellular components.

Histologically normal thymus (type A) in patients with myasthenia gravis (MG) was immunohistochemically compared with hyperplastic MG thymus (type B) and normal non-MG thymus. In formalin-fixed, paraffin-embedded sections of ten type A, ten type B, and eight non-MG cases, the thymic epithelium and other cellular components were stained in conjunction with the basement membrane by a double immunoenzymatic method. This technique demonstrated a moderate architectural disturbance in type A thymus, with distended perivascular space (PVS), elongated medullary epithelium, and disrupted basement membrane. These changes were more prominent in type B thymus but were minimal to lacking in non-MG thymus. Compared with those in non-MG thymus, the myoid cells in MG thymuses of both types tended to cluster around the Hassall's corpuscles, with a slight decrease in number in type B but not in type A. B-lymphocytes were present in type B, type A, and non-MG thymuses in that order of abundance; the cells were confined to the medullary parenchyma in the non-MG group but were numerous both in the PVS and medulla in the MG groups. T-lymphocytes were increased in the expanded PVS of type A and B MG thymuses. The number of interdigitating reticulum cells was similar in the three groups, but the cellular distribution was more dispersed in MG thymuses of both types. These findings, although previously described in type B thymus, have not been well recognized in type A thymus. They support the view that a common abnormality (presumably chronic thymitis), differing in degree only, underlies MG thymuses regardless of the presence of follicular hyperplasia.

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat.

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 mu Ci/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-microns frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.

Characterization of the binding of Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) to glycosphingolipids, using a solid-phase overlay approach.

Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) were radiolabeled externally (125I) or metabolically (35S) and analyzed for their ability to bind glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells. Two binding properties were found and characterized in detail. (i) Both bacteria showed binding to lactosylceramide (LacCer) in a fashion similar to bacteria characterized earlier. The activity of free LacCer was dependent on the ceramide structure; species with 2-hydroxy fatty acid and/or a trihydroxy base were positive, while species with nonhydroxy fatty acid and a dihydroxy base were negative binders. Several glycolipids with internal lactose were active but only gangliotriaosylceramide and gangliotetraosylceramide were as active as free LacCer. The binding to these three species was half-maximal at about 200 ng of glycolipid and was not blocked by preincubation of bacteria with free lactose or lactose-bovine serum albumin. (ii) A. naeslundii, unlike A. viscosus, showed a superimposed binding concluded to be to terminal or internal GalNAc beta and equivalent to a lactose-inhibitable specificity previously analyzed by other workers. Terminal Gal beta was not recognized in several glycolipids, although free Gal and lactose were active as soluble inhibitors. The binding was half-maximal at about 10 ng of glycolipid. A glycolipid mixture prepared from a scraping of human buccal epithelium contained an active glycolipid with sites for both binding specificities.

Identification of sequences of chromosome 7 that are expressed in sweat gland epithelial cells.

This paper describes an approach that can be used to identify specifically expressed coding sequences in defined regions of genomic DNA. We developed this method to identify expressed sequences from chromosome 7 located at or near the cystic fibrosis (CF) locus. Radioactively labelled single-stranded cDNAs derived from sweat gland epithelial cells and from fibroblasts were used to screen a genomic library constructed from flow-sorted chromosomes. Differential screening of phage lifts with these two probes yielded 36 different DNA segments. By using somatic cell hybrids containing different portions of chromosome 7, four of the clones were mapped to the 7q31 region in which the CF locus is located. These four clones and two others that gave strong differential epithelial signals but that were not within 7q31 were studied further. Restriction fragment length polymorphisms (RFLPs) were identified for two of the DNA segments within 7q31 and used for linkage analysis using a panel of CF families. One DNA segment was assigned to a location centromeric to the met locus. The other marker did not show recombination with CF but was subsequently excluded from the CF region by physical mapping. Three of the six DNA segments were found to hybridize to various RNAs using the Northern technique and therefore contain portions of genes. One of the clones showed strong differential expression when epithelial tissues were compared to fibroblasts and may represent an epithelium-specific gene.

A marker for primary choroid plexus neoplasms.

Primary choroid plexus (CP) tumors are rare neoplasms that present in childhood or, less frequently, in adult life. The majority are benign and amenable to complete surgical excision, but occasionally more invasive variants are encountered. Although generally pathologically distinct, occasionally primary CP neoplasms may be difficult to distinguish from metastatic papillary carcinomas or papillary ependymomas. Conventional cytologic markers are not sufficiently specific to permit accurate diagnosis of primary CP tumors. The authors have reported that the CP is the unique site of synthesis within the brain of transthyretin (TTR, prealbumin), a transport protein for thyroxine and retinol. They therefore investigated the utility of TTR as a biochemical marker for CP tumors. They detected intense immunoreactivity for TTR at high dilutions of primary antiserum in the neoplastic epithelium of all of nine primary CP tumors (six papillomas and three carcinomas), but not in eight cellular or three papillary intracerebral ependymomas, meningiomas, oligodendrogliomas, astrocytomas, primary extracerebral papillary carcinomas (three thyroid, two breast) or five of six cerebral metastases from systemic papillary carcinomas. In one case of cerebral metastasis from papillary thyroid carcinoma, rare isolated immunoreactive cells were observed. Faint staining of the stromal-ependymal junction was seen in myxopapillary ependymomas of the filum terminale, which were otherwise nonreactive. By in situ hybridization, TTR mRNA was abundant in neoplastic CP epithelium, confirming local TTR synthesis. The authors conclude that TTR is synthesized by neoplastic CP epithelium and is an excellent marker for primary CP neoplasms.

Flow cytometric evaluation of epithelial ovarian cancer.

We investigated the prognostic significance of deoxyribonucleic acid content and proliferative activity of tumor cell populations as measured by flow cytometry of the tumor specimens from 115 women with epithelial ovarian cancer. Deoxyribonucleic acid aneuploidy was found in 87 of 115 (76%) of these cancers with a mean deoxyribonucleic acid index of 1.6 and S-phase fraction of 14.7%. The S-phase fraction of the 28 (24%) diploid tumors was 7.0%. Deoxyribonucleic acid ploidy was significantly correlated with survival. S-phase fraction was significantly correlated with ploidy, residual tumor, histology, grade, ascites, time to recurrence, and survival. Diploidy versus aneuploidy were the best discriminating values for deoxyribonucleic acid index and an S-phase fraction of greater or less than 18% for that parameter. Multivariate analysis revealed stage, S-phase fraction, residual tumor, and grade to be independently associated with time to recurrence, and stage, age, S-phase fraction, and largest metastases were factors associated with survival. Deoxyribonucleic acid ploidy did not significantly improve either model. These results suggest that abnormalities of deoxyribonucleic acid content and the proliferative activity of tumor cell populations are reflective of their biologic activity.

Immunolocalization of type VI collagen in developing and healing rabbit cornea.

We have localized type VI collagen in normal developing and corneal scar tissue. Indirect immunofluorescence showed that type VI collagen was distributed throughout the normal stroma and most of the scar. No fluorescence was detected along the posterior margin of the scar and in a retrocorneal membrane continuous with the scar. Since the corneal endothelium in rabbits contributes to the formation of scar tissue and retrocorneal membrane, our observations suggest that the endothelium does not synthesize type VI collagen. Indirect immunoelectron microscopy showed that type VI collagen was located abundantly between collagen fibrils as fine filamentous structures containing beads with a periodicity of 100 nm, consistent with published observations of other tissues. Because these filaments are more prominent when stained with ruthenium red, and predigestion of tissue with Chondroitinase ABC enhances binding of monoclonal antibody to type VI collagen, proteoglycans probably are associated with this collagen in the cornea. Ultrastructural observations supported by previous biochemical analyses show that the proportion of type VI collagen to fibrillar collagen is smaller in scar tissue compared with fetal cornea. The abundance of type VI collagen and its distribution and association with proteoglycans in rabbit corneal tissues suggest that this macromolecule plays a role in the tensile strength and transparency of the stroma.

A monoclonal antibody to a megakaryoblast-like cell line detects a protein found in blood cells and in the epithelial cell lining of various rat tissues.

Megakaryoblasts of bone marrow differentiate into megakaryocytes that in turn are the source of blood platelets. We have raised monoclonal antibodies to a megakaryoblast-like cell line derived from rat bone marrow (RPM cells). One antibody (Mab 213) and the corresponding antigen has been characterized by Western blotting and immunohistochemistry. Biosynthetic labeling with [35S]methionine showed that this antigen is synthesized by the RPM cells. In Western blots the antibody recognized proteins of about 90 kDa and 160 kDa in Triton extracts of RPM cells, whereas it recognized proteins of about 160 kDa and 200 kDa in Triton extracts of rat platelets and one of about 200 kDa in Triton extracts of various rat tissues (kidney, lung, intestine, and heart). By immunohistochemistry, the antigen was localized to the apical part of the epithelium lining certain parts of kidney tubuli, bronchi and large intestine.