Fact0r5 that 1nteract w1th the rat a16um1n pr0m0ter are pre5ent 60th 1n hepat0cyte5 and 0ther ce11 type5 L.E. 8a6155, R.5. Her65t, A.L. 8ennett, and J.E. Darne11, Jr.7he R0ckefe11er Un1ver51ty, New Y0rk, New Y0rk 10021 U5A 7he rat a16um1n pr0m0ter 1n5erted 1n aden0v1ru5 d1rect5 tran5cr1pt10n 1n human and r0dent hepat0ma ce115 and 1n r0dent hepat0cyte5 (Fr1edman et a1. 1986) and 8a6155 et a1. (1986) 6ut n0t 1n HeLa ce115 0r mye10ma ce115.7he nuc1e0t1de5 6etween -43 and -156 0f the RNA 5tart 51te 0f the rat a16um1n 9ene are re4u1red f0r th15 ce11-5pec1f1c expre5510n. Pr0te1n 61nd1n9 5tud1e5 (f00tpr1nt5, ex0nuc1ea5e 111 5t0p5, and 9e1 5h1ft5) a11 1nd1cate 5pec1f1c 1nteract10n 1n the -80 t0 -130 re910n 0f the 9ene w1th fact0r5 pre5ent 1n nuc1ear extract5 0f hepat0cyte5 and hepat0ma5, 6ut a150 fr0m extract5 0f 0ther ce115 that d0 n0t expre55 the a16um1n 9ene. 70 065erve a16um1n pr0m0ter 61nd1n9, a 5ma11er am0unt 0f extract 0f 11ver ce11 nuc1e1 wa5 re4u1red c0mpared t0 extract5 0f HeLa ce11 0r k1dney ce11 nuc1e1. 1n add1t10n, the var10u5 te5t5 0f DNA-pr0te1n 1nteract10n d1d n0t 91ve 4ua11tat1ve1y 1dent1ca1 re5u1t5 w1th extract5 fr0m d1fferent ce115. H0wever, 1t 5eem5 c1ear that fact0r5 are pre5ent 1n 5evera1 ce11 type5 where a16um1n 9ene5 are 1nact1ve that w111 61nd t0 th05e DNA 5e4uence5 dem0n5trated t0 6e nece55ary f0r ce11-5pec1f1c expre5510n 0f th15 9ene. 7he5e fact0r5 c0u1d e1ther 6e 51m11ar 6ut n0n1dent1ca1 fact0r5 0r the 5ame fact0r5 that are m0d1f1ed d1fferent1y 1n d1fferent ce11 type5.[Key W0rd5:7ran5cr1pt10na1 c0ntr01; DNA 61nd1n9 fact0r5 0r pr0te1n5; a16um1n pr0m0ter] Rece1ved Decem6er 31, 1986; rev15ed ver510n rece1ved and accepted Fe6ruary 12, 1987. DNA 5e4uence e1ement5 nece55ary f0r mamma11an 9ene expre5510n have 6een detected 60th near t0 and d15tant fr0m RNA 1n1t1at10n 51te5 f0r RNA p01ymera5e 11. A 1ar9e pr0p0rt10n 0f 9ene5, whether they are expre55ed 1n v1rtua11y a11 ce115 0r whether the1r expre5510n 15 11m1ted t0 0ne 0r a few ce11 type5, c0nta1n c0mm0n e1ement5 5uch a5 the 7A7A 0r CAA7 60xe5 0r 5P1 61nd1n9 51te5 that 9enera11y 0ccur w1th1n the f1r5t 100 nuc1e0t1de5 up5tream 0f RNA 5tart 51te5 (Dynan and 7j1an 1983; J0ne5 et a1. 1985; 5awad090 and R0eder 1985; 6rave5 et a1. 1986). 709ether, the5e DNA e1ement5 and the pr0te1n5 that 61nd t0 them are th0u9ht t0 c0nvey p051t1ve 519na15 f0r tran5cr1pt10n (5ee McKn19ht and 7j1an 1986). 1n add1-t10n t0 the5e c0mm0n re9u1at0ry e1ement5, DNA 5e-4uence5 that are w1th1n 100-300 nuc1e0t1de5 0f the RNA 5tart 51te and that are re4u1red f0r ce11-5pec1f1c act1v1ty have 6een 1dent1f1ed 1n 9ene5 that are expre55ed 0n1y 0r ma1n1y 1n 11ver, pancrea5, 1en5, and 1ymph0cyte5, am0n9 a 9r0w1n9 num6er 0f add1t10na1 9ene5 that are act1ve 1n 0ther ce115 and t155ue5 (8anerj1 et a1. 1983; 61111e5 et a1. 1983; 0tt et a1. 1984; C1116ert0 et a1. 1985 Ed1und et a1. 1985; 0ver6eek et a1. 1985). 1n a few ca5e5, 5e4uence5 5evera1 k1106a5e5 d15tant are kn0wn t0 p1ay a r01e 1n e1ther 9enera1 0r ce11-5pec1f1c 9ene expre5510n (60d60ut et a1. 1986; C05ta et a1. 1986). A150, pre5um-a6...

Abstract. Antigens specific to pericentral hepatocytes have been studied in adult mouse liver, during fetal development, and in cultured fetal hepatoblasts. Antibody reactive with glutamine synthetase stained all fetal liver cells but almost all cells lost this antigen after birth; only a single layer of pericentral cells retained it in adulthood. In contrast, monoclonal antibodies to major urinary protein (MUP) did not detect the antigen until •3 wk after birth, after which time the cells within 6-10 call diameters of the central veins were positive. Cultured fetal liver cells from embryos at 13 + 1 d of gestation were capable of differentiating in vitro to mimic events that would occur had the cells remained in the animal. About 10-20 % of the explanted cells grew into clusters of hepatocyte-like cells, all of which stained with albumin antibodies. MUP monoclonals were reactive with one-half of the differentiated fetal hepatocytes. Glutamine synthetase was present in all hepatocytes after several days in culture and gradually decreased and remained in only occasional cells, all of which also contained the MUP antigen.These findings suggest that a sequence of gene controis characterizes expression of specific genes in developing liver, and that differentiating fetal hepatoblasts are capable of undergoing similar patterns of gene activity in culture.T rIE adult liver is arranged in acini with the afferent blood supply from the portal vein and the portal artery entering branching vessels at the microscopic level deliver blood to capillary-sized vessels, termed sinusoids, which conduct blood between cords (or plates) of hepatocytes to empty into a central vein. Thus hepatocytes are divided into two zones or regions: periportal (close to the portal afferent circulation) or pericentral (close to the efferent central vein) (51). It is widely accepted that all hepatocytes in rodents and humans contain certain proteins (for example albumin), while other proteins are found at higher levels in cells of one or the other zone (4,8,23,24,32,33,42,55,58,59), perhaps related to the higher nutrient, hormone, and/or oxygen content of blood in periportal compared with the pericentral region. However recent work using immunohistochemical labeling shows a distinct pericentral location for a number of different detoxification enzymes whose regulation is not obviously related to nutrient or oxygen supply (2, 52, 62). These antigens are present either exclusively or predominantly in the 6-10 cell layers immediately surrounding the central vein in adult rodents. In addition, antibodies to glutamine synthetase show this enzyme to be present in rats in a single layer of pericentral cells (22). To supplement our studies on gene expression in mouse hepatocytes, which have emphasized the importance of cell surface contacts in maintaining hepatocyte specific transcription (12), and to begin to examine hepatocytes during fetal development, we prepared a series of mAbs against mouse liver tissue. Many of the first group ofmAbs we obtained rec...

Rapid, convenient methods for monoclonal antibody (mAb) isolation are critical for determining the concentrations of therapeutic mAbs in human serum. This work uses porous nylon membranes modified with a HER2 peptide mimotope, KGSGS­GSQLG­PYELW­ELSH (KH19), for rapid affinity capture of Herceptin, a mAb used to treat breast cancer. Covalent linking of KH19 to poly­(acrylic acid)-containing films in porous nylon leads to a Herceptin-binding capacity of 10 mg per mL of membrane and allows selective Herceptin capture from diluted (1:3) human serum in 5 min. Liquid chromatography-mass spectrometry demonstrates the high purity of eluted Herceptin. Moreover, the fluorescence intensity of the protein eluted from membranes increases linearly with the amount of Herceptin spiked in loading solutions containing diluted (1:3) human serum. These results demonstrate the promise of mimotope-modified membranes for Herceptin analysis that does not require secondary antibodies or derivatization with fluorescent labels. Thus, mimotope-containing membranes may form part of a simple benchtop analysis system for assessing the concentrations of therapeutic mAbs.

As tested by DNase I digestion, the chromatin structure in several regions 5' to the rat serum albumin gene varies in tissues and cell lines that differ in transcription rate of this gene. Three DNase I-hypersensitive regions were found in hepatocyte nuclei but not in kidney cell nuclei. The sites were "2.8 kbp (site 1), 0.2 kbp (site 2), and 0.05 kbp (site 3) upstream from the cap site of the gene. In rat fetal liver tissue and rat hepatoma cell lines (FaO, C2, and C2-rev7), as well as in cultured primary hepatocytes where the rate of albumin gene transcription is lower tanm in adult liver, hypersensitive site (HSS) 1 was absent while sites 2 and 3 were present. In addition, the C2 cell line, which does not express albumin mRNA, contains a different HSS at position -1.5 kbp. Factors (proteins) bound to sites 2 and 3 may allow cell-specific transcription, but the additional factor interaction at site 1 could be required for a maximal rate of albumin gene transcription.