Microtubules are required for cytokeratin aggresome (Mallory body) formation in hepatocytes: an in vitro study.

Mallory bodies are cytokeratin-ubiquitin aggresomes that form in hepatocytes in many different chronic liver diseases. One of the key components in aggresome formation, not yet investigated in Mallory body formation, is the role of microtubules. An in vitro tissue culture assay is required to test for microtubule involvement in Mallory body formation so that Mallory body formation can be observed in the presence or absence of microtubule-disrupting agents. In this report, a new model of in vitro Mallory body formation was developed, which uses cultured hepatocytes isolated from drug-primed mice. When hepatocytes were incubated in the presence of antimicrotubule agents, they failed to form Mallory bodies. It is concluded that intact microtubules are required for Mallory body formation.

Heat shock proteins are present in mallory bodies (cytokeratin aggresomes) in human liver biopsy specimens.

Mallory bodies (MBs) are aggresomes, composed of cytokeratin and various other proteins, which form in diseased liver because of disruption in the ubiquitin-proteasome protein degradation pathway. Heat shock proteins (hsp's) are thought to be involved in this process because it was discovered that MB formation is induced by heat shock in drug-primed mice. It has been reported that ubiquitin and a mutant form of ubiquitin (UBB(+1)) are found in aggresomes formed in the neurons in Alzheimer's disease and in the liver MBs in various liver diseases. In addition, hsp 70 has been found in aggresomes in Alzheimer's and in MBs in drug-primed mice. Therefore, we hypothesized that hsp's might be involved in MB formation in human liver diseases. Liver biopsy sections were double-stained using ubiquitin and hsp 70 or 90b antibodies. Both hsps 70 and 90b were found in MBs in all liver diseases investigated including primary billiary cirrhosis, nonalcoholic steatohepatitis, hepatitis B and C, idiopathic cirrhosis, alcoholic hepatitis, and hepatocellular carcinoma. Ubiquitin and the hsp's colocalized in all MBs in the diseased liver sections. These results indicate that hsp involvement in MB formation is similar to that seen in aggresome formation in other conformational diseases.

The Mallory body as an aggresome: in vitro studies.

Prior in vivo studies supported the concept that Mallory bodies (MBs) are aggresomes of cytokeratins 8 and 18. However, to test this hypothesis an in vitro model is needed to study the dynamics of MB formation. Such a study is difficult because MBs have never been induced in tissue culture. Therefore, MBs were first induced in vivo in drug-primed mice and then primary cultures of hepatocytes from these mice were studied. Two approaches were utilized: 1. Primary cultures were transfected with plasmids containing the sequence for cytokeratin 18 (CK 18) tagged with green fluorescent protein (GFP). 2. Immunofluorescent staining was used to localize the ubiquitin-proteasome pathway components involved in MB-aggresome complex formation in primary hepatocyte cultures. The cells were double stained with a ubiquitin antibody and one of the following antibodies: CK 8, CK 18, tubulin, mutant ubiquitin (UBB+1), transglutaminase, phosphothreonine, and the 20S and 26S proteasome subunits P25 and Tbp7, respectively. In the first approach, fluorescence was observed in keratin filaments and MBs 48 h after the cells were transfected with the CK 18 GFP plasmid. Nascent cytokeratin 18 was preferentially concentrated in MBs. Less fluorescence was observed in the normal keratin filaments. This indicated that MBs continued to form in vitro. The immunofluorescent staining of the hepatocytes showed that CK 8 and 18, ubiquitin, mutant ubiquitin (UBB+1), P25, Tbp7, phosphothreonine, tubulin, and transglutaminase were all located at the border or the interior of the MB. These results support the concept that MBs are aggresomes of CK 8 and CK18 and are a result of inhibition of the ubiquitin-proteasome pathway of protein degradation possibly caused by UBB+1.

Aggresome formation in liver cells in response to different toxic mechanisms: role of the ubiquitin-proteasome pathway and the frameshift mutant of ubiquitin.

Aggresomes form in cells when intracellular proteins undergo conformational changes, as in so-called conformational diseases. This phenomenon has been observed in the liver and brain and in cell culture in response to abnormal protein formation, such as mutant proteins. In the case of the brain the frameshift mutant ubiquitin (UBB+1) is involved. Mallory body formation in the liver is one example of this phenomenon in vivo. Mallory body formation is common in a variety of liver diseases of diverse pathogenesis. The study of the Mallory body forming model indicated that drug-conditioned hepatocytes form Mallory bodies when mice are given colchicine, ethanol, okadaic acid, or exposure to heat shock. These findings suggest that aggresome formation is a common pathway of liver injury due to diverse mechanisms. To further characterize the role of this common pathway, drug-primed mice were exposed to different types of liver injury, i.e., using such drugs as thioacetamide, galactosamine, tautomycin, and the proteasome inhibitor PS341. Mallory body formation was induced by treatment with all the toxins tested, giving credence to the proposal that aggresome formation in the liver is a common pathway in response to different primary mechanisms of liver injury. The frameshift mutant UBB+1 was invariably found to colocalize with ubiquitin in the Mallory body, indicating its essential involvement in the mechanism of MB formation.

Expression of insulin-like growth factor binding proteins and collagen in experimental colitis in rats.

OBJECTIVES AND DESIGN: Crohn's disease is complicated by smooth muscle hyperplasia and stricture formation. Insulin-like growth factor (IGF)-1 and insulin-like growth factor binding proteins (IGFBPs) may be involved in stimulating intestinal smooth muscle growth and collagen synthesis. Therefore, we investigated the expression of IGFBPs, collagen and collagenase activity in rat colitis and the effects of IGF-1 on IGFBP and collagen expression in rat colonic smooth muscle cells. METHODS: Animals were sacrificed during a 4-week time course of 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis. RNA from the animals' colons was blotted and hybridized with collagen-1 and IGFBP mRNA probes. Tissue proteins were screened for IGFBPs by Western ligand blotting. Collagenase activity was measured by zymography. Rat colonic smooth muscle cells in primary culture were incubated with IGF-1 then collagen-1, and IGFBP mRNAs and proteins were measured. RESULTS: In the rat tissue, IGFBP-3 mRNA and protein were increased 2 h after induction of colitis. IGFBP-4 mRNA was elevated after 2 h and IGFBP-4 protein after 4 h. IGFBP-5 mRNA was upregulated after 2 h with a peak at 12 h. IGFBP-5 protein was upregulated after 1 h and reached a peak at 3 days. Collagen-1 mRNA was increased after 5 days. Collagenase levels were decreased after 1 h and returned to normal by 28 days. In rat colonic smooth muscle cells, IGF-1 increased collagen-1 and IGFBP-5 expression. CONCLUSION: We demonstrated an upregulation of IGFBP and collagen expression and a downregulation of collagenase in rat colitis. In colonic smooth muscle cells, we found an upregulation of collagen-1 and IGFBP-5 following IGF-1 incubation. These results suggest an important role of IGF-1 in the collagen synthesis in colitis, mediated by IGFBPs.

Mycophenolate mofetil impairs healing of left-sided colon anastomoses.

INTRODUCTION: Inadequate healing and consequent leakage from bowel anastomoses are a significant cause of postoperative morbidity and mortality. Immunosuppressive drugs are known to disturb healing processes and to impair the mechanical stability of bowel anastomosis. Mycophenolate mofetil (MMF) is an immunosuppressive agent that selectively inhibits the proliferation of T and B lymphocytes and has been shown to be effective in preventing allograft rejection after organ transplantation. Adverse effects are few; however, nothing is known in regard to possible adverse effects of MMF administration on the healing of bowel anastomosis. The aim of the present study was to evaluate the effect of systemic MMF administration on the healing of colon anastomoses in rats. METHODS: Rats underwent laparotomy, division of the left colon, and sigmoidostomy. MMF (25 mg/kg) or vehicle was administered intraperitoneally in two groups (n=21 per group) 3 days before surgery and then once daily until euthanization (7 animals per group; 2, 4, and 6 days after surgery). Bursting pressure measurements, histologic evaluation, morphometric analysis, mucin and collagen staining, and BrdU immunohistochemistry of the anastomotic site were performed. Furthermore, matrix protein expression at the anastomotic site was determined by collagen I and fibronectin Western blots. RESULTS: Administration of MMF significantly decreased anastomotic bursting pressure postoperatively. Accordingly, histology, mucin staining, and BrdU immunohistochemistry and measurements of the colonic crypt depth showed more extended inflammation, a significantly lower proliferation rate, and a significantly thinned mucosal layer in the MMF-treated groups when compared to control animals, whereas matrix synthesis at the anastomotic site was not different. CONCLUSIONS: The administration of the immunosuppressive agent MMF significantly impairs healing and mechanical stability of colon anastomosis in rats during the early postoperative period. MMF act to disturb host reparative processes mainly by impairment of reparative colonic epithelium proliferation and less by a disturbance of matrix synthesis.

Mycophenolate mofetil reduces tissue damage and inflammation in an experimental model of colitis in rats.

BACKGROUND: Lymphocytes are widely believed to be responsible for persistent intestinal inflammation in inflammatory bowel diseases. Mycophenolate mofetil (MMF) is a potent immunosuppressant that inhibits lymphocyte proliferation and has been shown to be effective in preventing allograft rejection after organ transplantation. The purpose of this study was to assess the modulating effects of MMF on intestinal inflammation in an experimental model of colitis in rats. METHODS: Colitis was induced by rectal instillation of trinitrobenzenesulfonic acid (TNBS) in ethanol in male Sprague-Dawley rats. One group of rats (n = 10) was treated with MMF i.p. (25 mg/kg b.w.) daily for 1 week starting 24 h after induction of colitis. A second group of rats (n = 10) was treated with MMF at the same dose 2 days, I day and 1 h prior to induction of colitis. Control animals (n = 10) received vehicle only. After being killed, colonic tissue was macroscopically evaluated for necrosis and microscopically for ulcerations. Sections were stained and examined for the presence of granulocytes. RESULTS: Administration of MMF after induction of TNBS colitis reduced macroscopic injury by 62% compared to control animals (P = 0.01). Microscopic ulcerations were reduced by 64% compared to controls (P = 0.009). In addition, posttreatment significantly reduced the number of granulocytes. MMF pretreatment did not significantly prevent macroscopic or microscopic tissue damage, or change the number of granulocytes. CONCLUSION: Systemic administration of MMF significantly ameliorates tissue damage in a model of experimental colitis in rats suggesting that this compound may play an important role as an immunosuppressant in the therapy of inflammatory bowel diseases.

Gender, power, and population change.

PIP: This report describes fertility and mortality trends in developing countries and discusses how gender is defined and measured in some countries. The discussion relies on case studies and country statistics to reveal how gender shapes the lives of all people in all societies. Gender is defined as the different roles women and men play in society. Gender is manifested in institutional structures, power relations, and culturally determined behavior. In no society do women and men share equal roles. The effects of inequality for women are manifested differently between countries. The 1994 International Conference on Population and Development in Cairo established the goal of gender equality. Educational enrollment and illiteracy are two measures of gender inequality that affect opportunities in society for advancement, power, and status. Girls are less likely to be enrolled in school than boys and more likely to have higher absenteeism rates. In China, absenteeism of girls is actually increasing under reforms. Marriage practices may devalue the investment in girls' education. Women experience different working conditions: they work longer hours, are paid less or not at all, and hold lower-status jobs. The exceptions are found in the Philippines and Brazil, where women hold more professional jobs than men. Women carry multiple responsibilities that consume time and prevent greater involvement in public life. Dowry and brideprice can constrain family relations. Women generally have fewer inheritance rights. Few women hold high-level public office positions or parliamentary seats. The extent to which gender inequality is reflected in demographic processes depends upon the gap in power in education, employment, and income. The relationship between gender and demographic processes is a central topic currently being researched.^ieng

China's "missing girls": prospects and policy.

PIP: Women's conditions in China are viewed as deteriorating. The reduction in government control over urban factories and other places of employment has resulted in fewer women being hired. This leaves women in positions with low wages, poor working conditions, and discrimination based on sex, age, and marital status. The shift from rural state-controlled agricultural collectives to patriarchal family controls resulted in land distribution that gave men preference. The rapid growth in the economy and higher standards of living placed women at risk. Although the pressures to kill girl babies have relaxed, there is growing interest in making money from abducting and selling women for prostitution, marriage, and slavery. Women gained in recent decades greater participation in the labor force and higher educational levels. Women's access to health care is better. Female life expectancy is 72 years compared to 69 years for men. Maternal mortality is 95 deaths per 100,000 live births. Yet women hold subordinate positions to men in their jobs, and women are segregated in the textile and service industries. Women in rural areas have primarily access to agricultural jobs, which can be combined with child care. Women are less likely to be promoted and are retired 5 years earlier than men. Women carry a double burden of domestic labor and paid or unpaid labor. Women still have higher illiteracy rates than men, and the gender gap in higher education has remained stable since the 1970s. The imbalanced sex ratio is increasing. In some provinces it is 114 males for every 100 females, when the normal ratio is 105-106 males per 100 females. An estimated 12% of females are unaccounted for each year. The reasons for the missing children are identified as sex-selective abortion, infanticide, or underreporting. The next five-year plan holds the hope for improvement in women's status and an end to abuses against women and children.^ieng

Role of insulin and insulin-like growth factor receptors in regulation of T84 cell monolayer permeability.

We previously showed that insulin and insulin-like growth factor (IGF)-I and IGF-II caused a dose-dependent increase in permeability through the paracellular pathway of T84 cell monolayers over 3-4 days. Here we have determined which cell surface receptors were involved in this response. Using radioligand binding studies and receptor cross-linking studies, we found that T84 cells possess insulin and IGF-I receptors. There were approximately 20 x 10(3) insulin receptors/cell with a dissociation constant (KD) of 0.5 nM and 29 x 10(3) IGF-I receptors with a KD of 0.6 nM for IGF-I. Cross-linking studies identified the alpha-subunit of insulin and IGF-I receptors with deduced molecular weights of 126 x 10(3) and 128 x 10(3), respectively. IGF-II bound to T84 cells with an apparent KD of approximately 2.0 nM. Radioreceptor cross-linking indicated that IGF-II interacted principally with the IGF-I receptor, although low levels of the IGF-II/mannose 6-phosphate receptor were also expressed on the cell surface. We then correlated the biological effect with the radioligand binding studies. It was first demonstrated that insulin and IGF-I were degraded in medium in the presence of cells. In addition, we showed that continuous exposure for 2-3 days to insulin or IGF-I was required to produce their biological effect on permeability. Taking into account the rate of degradation and the requirement for continuous exposure, we found a close correlation between radioligand binding and the half-maximal effective concentration for the hormonal effects on transepithelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of growth factor and cytokine-induced increases in T84 cell monolayer permeability by media components.

Previous studies have shown that insulin, insulin-like growth factors, and interferon-gamma cause an increase the paracellular permeability across T84 human colonic epithelial cell monolayers. The striking similarity in the time course and the structural changes in the actin cytoskeleton prompted us to test whether these factors influenced the paracellular permeability by a common mechanism. T84 cell monolayers were grown in variously modified media, and the effect of all three factors was tested by measuring the conductance of monolayers mounted in Ussing chambers. Media additions or deletions that modify cellular carbohydrate metabolism markedly influenced the response to all these peptides. In particular, the increase in conductance produced by each of the three peptides was 1) inhibited by addition of short-chain fatty acids, principally n-butyrate and propionate; 2) attenuated by the replacement of media glucose with slowly metabolized and nonmetabolized sugars; 3) potentiated by decreasing the media iron concentration; and 4) inhibited by increasing the media iron concentration. The effects of short-chain fatty acids, glucose, and iron were all shown to be dose dependent. In addition, the ability of high media iron to prevent the increase in permeability caused by all three factors was shown to require ambient oxygen. Together, these results strongly suggest that insulin, insulin-like growth factors, and interferon-gamma increase the permeability across T84 cell monolayers through a common mechanism that is modulated by glucose-derived metabolites and oxidative metabolism.

Regulation of T84 cell monolayer permeability by insulin-like growth factors.

When grown on permeable supports, the T84 human colonic epithelial cell line forms polarized monolayer cultures with high-resistance tight junctions between adjacent cells. Addition of either insulin-like growth factor (IGF) I or II to the basolateral but not the apical membrane side of established monolayers caused a dose-dependent decrease in transepithelial resistance over a 4-day period. IGF-I was more potent than IGF-II, with half-maximally effective concentrations of 0.7 and 2.2 nM, respectively. Both IGF-I and -II caused a parallel increase in the transepithelial flux rates for Na+ and the extracellular space marker, mannitol, demonstrating that the decrease in electrical resistance was due to increased permeability through the tight junction-regulated paracellular pathway. Simultaneous addition of cycloheximide prevented the decline in electrical resistance, implying that protein synthesis is necessary for the effect of IGF on paracellular permeability. Treatment of monolayers with IGF produced a subtle condensation of the perijunctional actin ring as visualized using rhodamine-labeled phalloidin. These results demonstrate that IGF-I and -II regulate the paracellular permeability of T84 cell monolayers through a receptor-mediated process that probably involves changes in protein synthesis and cytoskeletal structure.

Recent trends in fertility and nuptiality in china.

The State Family Planning Commission of China has conducted two large-scale fertility surveys, in 1982 and 1988, covering sample households containing 1 million and 2 million persons, respectively. These surveys obtained lifetime histories, including age at first marriage and at each birth for female members of the households from ages 15 to 67 in the first survey and from 15 to 57 in the second. The data provide detailed information on the extraordinary decline in the rate of childbearing in China (by 60% from 1970 to 1980). Because rising age at marriage played a significant role in this decline, the effect of changes in the pattern of entry into marriage on childbearing since 1980 was examined. There was a sharp increase in overall fertility (the total fertility rate) from 1980 to 1982; after falling to slightly below the 1980 level in 1985, the rate rose in 1985 and 1986 to well above that of 1980. A major factor in this arrested and partially reversed decline was a boom in marriage that followed a relaxation in 1980 of locally administered restrictions on marriage before the officially designated desirable age. In fact, the total fertility rate of married women (summed over duration of marriage rather than age) averaged much lower in the mid-1980s than in 1980. The summary rate of bearing second children increased markedly in the 1980s when calculated by age of women, but declined when calculated by duration of marriage, given the inflated number of recently married women.