Stem cell biology, hormone/matrix synergies and liver differentiation.

Extracellular matrix, in conjunction with hormones and growth factors, plays a profound role in regulating gene expression in liver and in all tissues. The availability of matrix components for liver cells in culture is having a major impact on our ability to study every aspect of gene expression. Its significance has been particularly profound in permitting cultures with partial restoration of transcription rates of tissue-specific mRNAs. However, the matrix reagents available to date are not so potent as the forms known to be present uniquely in the liver. Thus, there is a great need to purify and characterize these tissue-specific matrix components (e.g. heparin-PG) to enable the eventual production of them for general use by investigators. The use of matrix reagents and the eventual use of cultures of liver stem cells will begin a new era in the understanding of liver differentiation.

Mechanism of rejection of virus persistently infected tumor cells by athymic nude mice.

Cell lines known to be tumorigenic in the nude mouse were modified by rendering them persistently infected (P.I.) with a variety of RNA viruses, including measles, mumps, vesicular stomatitis virus, and influenza. Although as few as 100 HeLa or BHK cells produced tumors in 100% of nude mice, as many as 2 x 10(7) of the same cells P.I. with viruses failed to produce tumors. An active host response responsible for restricting the growth of the P.I. cells was suggested by the findings of marked mononuclear cell infiltrates at the inoculation sites and the inability of irradiated nude mice to reject them. An analysis of the in vitro cytotoxic activity of spleen cells from normal nude mice indicated that: (a) P.I. cell lines, but not uninfected cell lines, were susceptible to spontaneous cytotoxicity; (b) in vivo inoculation of P.I. lines induced an enhanced cytotoxic activity for P.I. targets in vitro, and this induction was not specific either for inducing virus or cell line; and (c) the effector cell had the characteristics for natural killer (NK) cells. Although the specificity of recognition of the various P.I. cell lines remains unclear, cold competition experiments indicated that blocking the killing of one P.I. cell line, e.g. HeLa-measles, could be achieved only by unlabeled homologous cells, i.e. HeLa-measles, and not by uninfected cells or other P.I. lines. A variant subline of BHK cells P.I. with VSV was selected for its ability to withstand the rejection process in nude mice. These cells formed metastatic and invasive tumors in nude mice. Although they were the most potent inducers in vivo of NK cell activity against various P.I. targets, they were the most resistant of the P.I. lines to NK cell cytotoxicity in vitro. In this system there was a good correlation between tumor rejection in vivo and susceptibility to NK cells in vitro. The present results suggest that NK cells may play a significant role in both rejection of tumor cells, and in resistance to viruses, particularly persistent infections.

Connective tissue biomatrix: its isolation and utilization for long-term cultures of normal rat hepatocytes.

A new procedure is introduced for the isolation of connective tissue fibers, called biomatrix, containing a significant portion of the extracellular matrix (basement membrane components and components of the ground substance). Biomatrix isolated from normal rat liver contains >90% of the tissue's collagens and all of the known collagen types, including types I and III and basement membrane collagens. The purified collagenous fibers are associated with noncollagenous acidic proteins (including fibronectins and possibly small amounts of glycosaminoglycans). Procedures are also described for preparing tissue culture substrates with these fibers by either smearing tissue culture dishes with frozen sections or by shredding the biomatrix into small fibrils with a homogenizer. The biomatrix as a substrate has a remarkable ability to sustain normal rat hepatocytes long-term in culture. The hepatocytes, which on tissue culture plastic or on type I collagen gels do not survive more than a few weeks, have been maintained for more than 5 mo in vitro when cultured on biomatrix. These cells cultured on rat liver biomatrix show increased attachment and survival efficiencies, long-term survival (months) and retention of some hepatocyte-specific functions.

Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures.

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata.

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan real-time PCR assay targeting the cpsD gene.

AIMS: The development and evaluation of a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. METHODS AND RESULTS: A TaqMan-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan PCR assay detected 1 pg of purified DNA and 10(4)P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92.0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. CONCLUSIONS: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. SIGNIFICANCE AND IMPACT OF THE STUDY: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.

Role in nude mice of interferon and natural killer cells in inhibiting the tumorigenicity of human hepatocellular carcinoma cells infected with hepatitis B virus.

The human hepatoma cell line, PLC/PRF/5, which is persistently infected with hepatitis B virus (HBV), has integrated HBV-DNA, secretes HBV surface antigen (HBsAg), and does not grow readily in congenitally athymic (nu/nu) mice. The present investigation was undertaken to ascertain whether the low tumorigenicity of this cell line was governed by a host immune response and/or was related to expression of HBsAg. Subcutaneous injection of 4-5 X 10(6) cells into BALB/c nude mice produced localized encapsulated tumors with morphologic features of primary hepatocellular carcinoma in 25% of the animals within 29-40 d. No tumor growth was observed at lower cell inocula. In contrast, SK-HEP-1, an HBV-negative human hepatoma cell line, produced tumors at 1-5 X 10(6) cells inocula in 66% of the animals. Immunosuppression of mice with antilymphocyte serum (ALS) or irradiation increased tumor incidence in mice inoculated with 1 X 10(6) PLC/PRF/5 cells to almost 100% and produced local invasiveness. Immunosuppression also reduced the latency, i.e., time to tumor appearance, and increased mean tumor weight. These results suggest that tumorigenicity was limited by the host immune response. The nature of the response was delineated by treating nude mice challenged with tumor cells with sheep anti-mouse interferon globulin (anti-IFN). When 2 X 10(6) cells were injected, tumor growth occurred in 75% of anti-IFN-treated mice, whereas controls injected with the same number of cells, but not receiving anti-IFN, failed to develop tumors. The tumors in the anti-IFN-treated mice were highly invasive and the latency period until tumor appearance was reduced to 3-5 d. An inverse correlation was found between susceptibility of the hepatoma cells to natural killer (NK) activity in vitro and resistance to tumor growth in vivo. In vitro cytotoxicity for PLC/PRF/5 cells was eliminated by anti-NK 1.1 and complement, establishing the effector cell as an NK cell. NK cell activity 14 d after inoculation of mice with PLC/PRF/5 cells was augmented against PLC/PRF/5 target cells but not against SK-HEP-1 cells. Treatment of mice with ALS, irradiation, or anti-IFN abolished NK activity against PLC/PRF/5 cells. Co-cultivation of nude mouse spleen cells with PLC/PRF/5 but not with HBsAg or SK-HEP-1 cells induced secretion of murine IFNalpha. These results suggest that the IFN/NK cell system may play a role in limiting tumorigenicity and invasiveness of HBV-infected human hepatocellular carcinoma cells by a mechanism similar to that found for other cells persistently infected with viruses.

Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: relevance to clonal growth of tumors.

Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells. Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-1 and PLC/PRF/5. Heparin's inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF beta). HepG2 cells expressed high basal levels of mRNAs encoding IGF II and TGF beta that were inducible, through transcriptional and posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II and TGF beta, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects of insulin, glucagon, and growth hormone in combination with heparin; TGF beta was regulated primarily by the synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations regulated TGF beta posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-1 either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGF beta, whose mRNA synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and the biological responses to them.

Regulation of insulin mRNA abundance and adenylation: dependence on hormones and matrix substrata.

The insulin mRNA levels of rat insulinoma cell lines increased six- to eightfold, and the cells entered a transient state of growth arrest when they were cultured in serum-free, hormonally defined medium and on an extract of extracellular matrix derived from a basement membrane-secreting tumor line, EHS. Insulinoma cultures in growth arrest responded to glucose with a two- to threefold increase in insulin secretion associated with an insulin mRNA that contained a poly(A) tail that was 120 to 140 bases longer than normal.

Posttranscriptional modulation of gene expression in cultured rat hepatocytes.

The maintenance of high levels of two liver-specific mRNAs in cultured hepatocytes was achieved in a serum-free hormonally defined cell culture medium. However, this maintenance of liver-specific mRNA levels did not correlate with the level of transcription of the genes but was apparently due to increased stabilization of the tissue-specific mRNAs. The mRNA stabilization did not occur in serum-supplemented medium. In both defined and serum-supplemented medium, actin and tubulin mRNAs were also greatly increased, in both cases predominantly if not entirely due to increased mRNA stability.

Negative and positive cis-acting elements control the expression of murine alpha 1-protease inhibitor genes.

The alpha 1-protease inhibitor (alpha 1-PI) proteins of mice are encoded by a group of genes whose members are expressed coordinately in a liver-abundant pattern and are regulated primarily at the transcriptional level. To better understand the developmental and tissue-specific regulation of this gene family, one member that is analogous to the human alpha 1-antitrypsin gene was chosen for study. Deletional analysis of the upstream regulatory region of this gene was performed, spanning from -10 kilobases to -80 base pairs relative to the transcriptional start site. Two functional positive cis-acting elements within the 522 bases immediately upstream of the start site for transcription were shown to modulate the level of expression from this promoter when introduced into human or mouse hepatoma cells, and a third region acted as a negative regulatory element in that its deletion resulted in a two- to sixfold increase of expression of a transfected minigene construct. Sequence comparison between the regulatory domains of two mouse alpha 1-PI genes and the human alpha 1-antitrypsin gene showed that the mouse gene contains a novel positive cis-acting element which is absent in human gene and that a specific eight-base-pair difference between species results in a strong positive cis-acting element in the human gene acting as a negative element in the mouse gene. An enhancer located approximately 3,000 base pairs upstream of the major start site for transcription was also identified. This element is position and orientation independent. Several different DNA-protein binding assays were used to demonstrate that each DNA segment with functional significance in transfection assays interacts specifically with proteins found in adult mouse liver nuclei. The major positive-acting element appeared to be specifically recognized by nuclear proteins found only in tissues that express alpha 1-PI, while the negative element binding proteins were ubiquitous. Thus, the distal regulatory domain including bases -3500 to -133 of this murine alpha 1-PI gene family member is more complex than was previously demonstrated. It is composed of a set of at least three additional functional cis-acting regulatory elements besides those which have been mapped by others and has a far upstream enhancer.

Growth of human breast carcinomas in nude mice and subsequent establishment in tissue culture.

Thirty-two malignant human breast tumors were implanted s.c. in female nude mice. Seven tumors survived for two passages, and four were established into permanent transplantable tumor lines. The transplantable tumors have retained the histopathology of the original tumor throughout passaging in the nude mice. In addition, two of the transplantable tumors have low concentrations of estrogen receptor. Tissue culture of the original tumor specimens upon receipt resulted in epithelial outgrowth in 15 of 32 primary cultures. However, no permanent cell lines were established. Attempts to culture 23 tumors frozen with dimethyl sulfoxide upon receipt were unsuccessful. In contrast, establishment of cell strains was successful with tumor specimens cultured following passage in the nude mice; three cell strains were initiated from two of the transplantable tumors.

Suramin inhibits growth and yet promotes insulin-like growth factor II expression in HepG2 cells.

Suramin, a drug shown to inhibit the growth of some tumor cell types in vivo and in vitro, was found to strongly inhibit the proliferation of the human hepatoma cell line, HepG2, grown in either serum-supplemented medium or a serum-free, hormonally defined medium tailored for hepatoma cells. In parallel, suramin induced the expression of the 6.0-kilobase transcript of insulin-like growth factor II (IGF II) but had no significant effect on transforming growth factor beta 1 mRNA levels in these cells. The induction in the abundance of IGF II mRNA was posttranscriptionally regulated. The growth-inhibitory effect of suramin was not mediated through IGF II, since addition of IGF II directly to the medium mildly stimulated the growth of HepG2 cells in a dose-responsive manner. Treatment of cells with suramin for 24 h resulted also in increased albumin mRNA levels in both HepG2 cells and normal rat hepatocytes. Suramin's effect on albumin occurred only in cells in medium supplemented with serum and in freshly plated cells, i.e., cells that had not been in culture long enough to form their own extracellular matrix substratum. We hypothesize that, as for heparin, suramin can bind serum factor(s), adversely affecting the stability of albumin mRNA. Addition of either IGF I or IGF II directly to cells resulted in an increase in albumin mRNA in HepG2 cells after 4 days in culture, implicating a role for these factors in differentiation. Yet they showed no effect unless the cells were grown for 2 days in serum-supplemented medium and then switched to a hormonally defined medium. Thus, both mitogenic and differentiation effects of IGFs were observed, and the qualitative responses of the cells to IGFs were dictated by other variables. Neither suramin nor IGF II had an effect on total sulfation levels in cells or medium conditioned by HepG2 cells and rat hepatocytes, suggesting that they have few, if any, effects on glycosaminoglycan synthesis or the extent of sulfation. Therefore, at present, suramin's potent biological effects on the growth and differentiation of HepG2 cells and rat hepatocytes are clearly complex and mediated through as yet unclear mechanisms.

Clonal growth of tumors on tissue-specific biomatrices and correlation with organ site specificity of metastases.

We have found that neoplastic transformation alters the ability of cells to grow on substrata of tissue extracts, "biomatrices", enriched in extracellular matrix. Tumor cells were able to survive and grow at lower densities and on more types of biomatrices than normal cells. When plated at high densities (greater than 10(5) cells/60 mm dish), tumor cells attached with equal efficiency and grew at similar rates and to equivalent saturation densities on biomatrices derived from all tissues. However, at low (10(2)-10(4)/60-mm dish) seeding densities, the tumor cells grew only on certain types of biomatrix. For the various hepatoma and mammary carcinoma cell lines tested, the tissue specificity in clonal growth on biomatrices correlated with their organ site specificity for metastasis in vivo in immunosuppressed, athymic nude mice. Analysis of the effects of purified matrix components (adhesion proteins, collagens, glycosaminoglycans) indicated that only the glycosaminoglycans influenced density-dependent survival and growth of tumor cells with effects that differed with respect to the cell's metastatic potential. The results indicate that the ability of tumor cells to colonize specific tissues represents, in part, regulation of low density survival and growth by extracellular matrix and are suggestive that one of the matrix components responsible may be proteoglycans or their glycosaminoglycan chains.

Characterization of a human, sex steroid-responsive transitional cell carcinoma maintained as a tumor line (R198) in athymic nude mice.

We have established a transplantable tumor line, R198, derived from a papillary (transitional cell) carcinoma of the human urinary bladder. In nude mice, the tumor line exhibits properties attributable to both prostatic and transitional epithelia. In tumor-bearing animals given androgens, the neoplasm has a rapid growth rate, possesses low levels of measurable androgen receptors, produces tartrate-inhibitable acid phosphatase, and forms well-encapsulated, cystic tumors composed of transitional, glandular, and squamous cells. The administration of estrogens or transplantation of the tumor into female mice causes regression of the tumor. In a small percentage of the transplants placed into females or estrogenized animals, selection occurs for tumor cells which can grow under these conditions. The resulting tumors are infiltrating scirrhous carcinomas that closely resemble squamous cell carcinomas of the urinary bladder. These neoplasms grow slowly and do not possess androgen receptors or secretory material. They are composed of a homogeneous population of squamous cells which are locally invasive. The paradox of a bladder tumor with some prostatic characteristics may be explained by the fact that the tumor was derived from the trigone region of the bladder, which embryologically is formed by an admixture of tissue from the wolffian duct and the urogenital sinus. Some trigone-derived neoplasms have characteristics of both bladder and prostate. We hypothesize that sex steroid-sensitive R198, with characteristics of both bladder transitional cells and prostatic epithelia, is a tumor which phenotypically expresses the embryological origins of these tissues. As such, the tumor line will serve as a useful model for studying sex steroid-responsive cells of the urogenital epithelium.

Tumorigenicity in nude mice of a human hepatoma cell line containing hepatitis B virus DNA.

The human hepatocellular carcinoma cell line PLC/PRF/5, which synthesizes and secretes hepatitis B surface antigen, was grown under optimal conditions in tissue culture, using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 10(-11) M triiodothyronine on collagen rafts. Injection s.c. of the PLC/PRF/5 cell line into athymic BALB/c nude mice resulted in the growth of a well-circumscribed, moderately differentiated hepatocellular carcinoma. The intervals until tumor appearance and tumor "take" rates were dependent on inoculum dose. Four to 5 x 10(6) cells induced tumor growth in 29% of 14 injected mice within 29 to 40 days, while 7 to 13 X 10(6) cells induced tumors in all 15 mice within 10 to 12 days after inoculation. Hepatitis B surface antigen was detected in the nude mouse serum and tumor tissue, and its concentration roughly correlated with tumor weight. A low level of antibody against hepatitis B surface antigen was detected in five tumor-bearing animals, as well as in one mouse which did not produce a tumor. Hepatitis B core antigen and its antibody and hepatitis B e antigen and its antibody were not detected in 26 mice, using immunohistochemical and radioimmunoassay methods. alpha-Fetoprotein, carcinoembryonic antigen, and alpha-antitrypsin were detected in nude mice tumors, using the peroxidase-antiperoxidase technique. Finally, hepatitis B virus DNA, identified in the nude mouse tumor by molecular hybridization techniques, was compared to PLC/PRF/5 cell line hepatitis B virus DNA.

Congenital pulmonary vein stenosis: structural changes in a patient with normal pulmonary artery wedge pressure.

A male infant is described who died at 13 months of age with stenosis of all extrapulmonary veins except the left upper vein. The pulmonary artery wedge pressure was normal, the first time this is reported in this condition. At autopsy, there were structural changes of the pulmonary arteries and veins in all lobes with or without pulmonary vein stenosis. Arterial changes-muscle extension, medial hypertrophy and decreased arterial size--analyzed quantitatively were found to be similar in all lobes. Venous medial hypertrophy was more marked in obstructed lobes. These anatomic changes are presumably due to fixed venous obstruction in the pulmonary lobes drained by stenotic veins and to high flow in the left upper lobe.

Transcriptional and posttranscriptional control of connexin mRNAs in periportal and pericentral rat hepatocytes.

Distinct patterns of expression of gap junction, or connexin, mRNAs were observed in periportal vs. pericentral hepatocytes. The two cellular fractions (isolated from rat livers by perfusion) were more than 90% parenchymal, as determined by flow cytometry for a hepatocyte-specific marker. The periportal and pericentral fractions were identifiable due to enrichment in enzymatic activities previously shown to be differentially expressed in the respective regions of liver. Northern blot analyses revealed that mRNA encoding connexin 26 was 2.8 times more abundant in the periportal than in the pericentral cells, while connexin 32 mRNA was equally distributed. Messenger RNA from each fraction was radiolabeled in order to compare the relative abundance of the connexin mRNAs in each fraction. The ratio of connexin 26 to connexin 32 mRNA in the portal fraction was about 0.085, and in the central fraction about 0.038. Connexin 26 mRNA was transcribed, however, at a faster rate than connexin 32 mRNA by nuclei isolated from both cellular fractions. Connexin 26 mRNA was transcribed at 3.9 times the rate in nuclei from the periportal than from the pericentral cells. These data suggest that while the zonation of connexin 26 mRNA synthesis in liver appears to be controlled transcriptionally, posttranscriptional regulatory mechanisms determine the relative abundance of the connexin mRNAs.

Mucous membrane of respiratory epithelium.

Of the eight epithelial cell types of the airway surface epithelium three are secretory, the mucous, serous and Clara cells: the first two are also found in the submucosal glands. Organ culture results indicate that the pattern of control of the surface cells is different from that of the glands. Our recent studies show that in the surface epithelium Clara and serous cells can quickly convert to mucous as do nonsecretory undifferentiated cells. The balance between the various cell types changes with airway level. The type of glycoprotein within the secretory granules is neutral or acid, sialylated or sulfated, and also shows a regional pattern. Homeostasis is maintained in the normal but the equilibrium is quickly upset by a variety of irritants, infection of drugs. Change in pattern of glycoprotein synthesis depends largely on change of granules. The granules at the cell apex change first. The nature and distribution of the various receptor binding sites is significant in patterns of control. This lability occurs in an intact epithelium and whether or not mitotic activity is increased. With irritation tolerance to stimulus develops. Antiinflammatory agents can protect against some of these cellular and intracellular events. Our organ culture studies and biochemical analysis of secretion complement the tissue studies. Recent studies show that isoproterenol and salbutamol (a nonselective beta agonist and a selective beta 2, respectively) alter the normal mix of cell types and quickly, but that they have different regional specificity.

Down syndrome: patterns of disturbed lung growth.

Abnormal pulmonary development characterized by decreased alveolar complexity has been previously reported in patients with Down syndrome. We investigated the state of pulmonary development and found several undescribed patterns of disturbed lung growth. The axial branches of intrasegmental airways were counted in 13 Down syndrome patients; in nine, airway generation was reduced by 25% or more of the predicted number. The radial alveolar counts were evaluated in 11 lungs: five were 143% to 162% above expected (four to above adult values), five were as expected, and one was below expected (82%). No correlation was found between airway number and radial alveolar count. Our finding of the reduction in airway branching in the lungs of patients with Down syndrome suggests interference with development before birth. Disturbances in alveolar multiplication are also found.