Metabolic-associated fatty liver disease and hepatocellular carcinoma: a prospective study of characteristics and response to therapy.

BACKGROUND AND AIM: The rising incidence of hepatocellular carcinoma (HCC) in Australia is related to increasing rates of metabolic-associated fatty liver disease (MAFLD). This study aimed to prospectively characterize the metabolic profile, lifestyle, biometric features, and response to treatment of HCC patients in an Australian population. METHOD: Multicenter prospective cohort analysis of newly diagnosed HCC patients at six multidisciplinary team meetings over a 2-year period. RESULTS: Three hundred and thirteen (313) newly diagnosed HCC patients with MAFLD (n = 77), MAFLD plus other liver disease (n = 57) (the "mixed" group), and non-MAFLD (n = 179) were included in the study. Alcohol-associated liver disease (ALD) (43%) and MAFLD (43%) were the most common underlying liver diseases. MAFLD-HCC patients were older (73 years vs 67 years vs 63 years), more likely to be female (40% vs 14% vs 20%), less likely to have cirrhosis (69% vs 88% vs 85%), showed higher ECOG, and were less likely to be identified by screening (29% vs 53% vs 45%). Metabolic syndrome was more prevalent in the MAFLD and mixed groups. The severity of underlying liver disease and HCC characteristics were the same across groups. While the MAFLD population self-reported more sedentary lifestyles, reported dietary patterns were no different across the groups. Dyslipidemia was associated with tumor size, and those taking statins had a lower recurrence rate. CONCLUSION: Equal to ALD, MAFLD is now the most common underlying liver disease seen in HCC patients in Australia. Future HCC prevention screening and treatment strategies need to take this important group of patients into consideration.

Groundwater protection and unconventional gas extraction: the critical need for field-based hydrogeological research.

Unconventional natural gas extraction from tight sandstones, shales, and some coal-beds is typically accomplished by horizontal drilling and hydraulic fracturing that is necessary for economic development of these new hydrocarbon resources. Concerns have been raised regarding the potential for contamination of shallow groundwater by stray gases, formation waters, and fracturing chemicals associated with unconventional gas exploration. A lack of sound scientific hydrogeological field observations and a scarcity of published peer-reviewed articles on the effects of both conventional and unconventional oil and gas activities on shallow groundwater make it difficult to address these issues. Here, we discuss several case studies related to both conventional and unconventional oil and gas activities illustrating how under some circumstances stray or fugitive gas from deep gas-rich formations has migrated from the subsurface into shallow aquifers and how it has affected groundwater quality. Examples include impacts of uncemented well annuli in areas of historic drilling operations, effects related to poor cement bonding in both new and old hydrocarbon wells, and ineffective cementing practices. We also summarize studies describing how structural features influence the role of natural and induced fractures as contaminant fluid migration pathways. On the basis of these studies, we identify two areas where field-focused research is urgently needed to fill current science gaps related to unconventional gas extraction: (1) baseline geochemical mapping (with time series sampling from a sufficient network of groundwater monitoring wells) and (2) field testing of potential mechanisms and pathways by which hydrocarbon gases, reservoir fluids, and fracturing chemicals might potentially invade and contaminate useable groundwater.

Expression of genes involved in lipogenesis is not increased in patients with HCV genotype 3 in human liver.

Hepatitis C virus (HCV) infection is frequently associated with hepatic steatosis, particularly in patients with HCV genotype-3 (HCVGT3). It has variously been hypothesized, largely from in-vitro studies, to be the result of increased synthesis, decreased metabolism and export of triglycerides. We measured by real-time PCR the expression of genes involved in lipid metabolism [acetyl-Coenzyme A carboxylase alpha, apolipoprotein B (APOB), diacylglycerol O-acyltransferase 2, fatty acid-binding protein 1, fatty acid synthase, microsomal triglyceride transfer protein (MTTP), peroxisome proliferator-activated receptor alpha (PPARA), peroxisome proliferator-activated receptor gamma (PPARG), protein kinase AMP-activated alpha 1 catalytic subunit (PRKAA1) and sterol regulatory element-binding transcription factor 1 (SREBF1)] in liver biopsies from patients infected with HCV genotype-1 (HCVGT1), HCVGT3 and Hepatitis B (HBV) using ß-glucuronidase (GUSB) and splicing factor arginine/serine-rich 4 (SFRS4) as housekeeping genes. Patients infected with HCVGT3 were younger than those infected with HCVGT1 (36.3 ± 2.5 vs 45.6 ± 1.5, P < 0.05, Mann-Whitney) and were more likely to have steatosis (69.2%vs 11.8%). No significant difference was found in the expression of genes involved in lipogenesis or transport in patients infected with HBV or HCV of either genotype. Contrary to expectation, given the greater degree of steatosis in HCVGT3-infected liver, expression of enzymes involved in lipogenesis was not elevated in HCVGT3 compared with HCVGT1 or HBV-infected liver. Significantly less mRNA for SREBF1 was found in HCVGT3-infected liver tissue compared with HCVGT1-infected liver (1.00 ± 0.06 vs 0.70 ± 0.15 P < 0.05). These results suggest that steatosis in patients infected with HCVGT3 is not the result of a sustained SREBF1 driven increase in expression of genes involved in lipogenesis. In addition, a significant genotype-independent correlation was found between the expression of APOB, MTTP, PRKAA1 and PPARA, indicating that these networks are functional in HCV-infected liver.

Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody-drug conjugate SGN-75.

BACKGROUND: CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody-drug conjugate, SGN-75. RESULTS: We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo. CONCLUSION: Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.

In-well degassing issues for measurements of dissolved gases in groundwater.

Measurement of dissolved gases in groundwater is becoming increasingly common and important. Many of these measurements involve monitoring or sampling within wells or from water pumped from wells. We used total dissolved gas pressure (TDGP) sensors placed in the screened section of various wells (4 to 72 m deep) to assess the dissolved gas conditions for open wells compared to the conditions when sealed (i.e., isolated from the atmosphere) with a hydraulic packer (one well) or when pumped. When the packer was installed (non-pumping conditions), TDGP rose from <1.7 to >3.1 atm (<172 to >314 kPa), with declines noted when the packer was removed or deflated. While pumping, TDGP measured in many of the wells rose to substantially higher levels, up to 4.0 atm (408 kPa) in one case. Thus, when groundwater is gas charged, the background aquifer TDGP, and likewise the dissolved gas concentrations, may be substantially higher than initially measured in open wells, indicating significant in-well degassing. This raises concerns about past and current methods of measuring the dissolved gases in groundwater. Additional procedures that may be required to obtain representative measurements from wells include (1) installing in-well hydraulic packers to seal the well, or (2) pumping to bring in fresh groundwater. However, observed transient decreased TDGPs during pumping, believed to result from gas bubble formation induced by drawdown in the well below a critical pressure (relative to TDGP), may disrupt the measurements made during or after pumping. Thus, monitoring TDGP while pumping gas-charged wells is recommended.

CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers.

CD133/prominin-1 is a pentaspan transmembrane glycoprotein overexpressed in various solid tumours including colorectal and glioblastomas. CD133 was found here to be highly expressed in >or=50% of pancreatic, gastric and intrahepatic cholangiocarcinomas. Quantitative flow cytometric analysis showed that a panel of established hepatocellular, pancreatic and gastric cancer cell lines expressed CD133 at levels higher than normal epithelial cells or bone marrow progenitor cells. A murine anti-human CD133 antibody (AC133) conjugated to a potent cytotoxic drug, monomethyl auristatin F (MMAF), effectively inhibited the growth of Hep3B hepatocellular and KATO III gastric cancer cells in vitro with IC(50) values of 2-7 ng ml(-1). MMAF induced apoptosis in the cancer cells as measured by caspase activation. The anti-CD133-drug conjugate (AC133-vcMMAF) was shown to internalise and colocalised with the lysosomal marker CD107a in the sensitive cell lines. In contrast, in the resistant cell line Su.86.86, the conjugate internalised and colocalised with the caveolae marker, Cav-1. Addition of ammonium chloride, an inhibitor of lysosomal trafficking and processing, suppressed the cytotoxic effect of AC133-vcMMAF in both Hep3B and KATO III. Anti-CD133-drug conjugate treatment resulted in significant delay of Hep3B tumour growth in SCID mice. Anti-CD133 antibody-drug conjugates warrant further evaluation as a therapeutic strategy to eradicate CD133+ tumours.

Separation of physical loading from photosynthesis/respiration processes in rivers by mass balance.

Diurnal variations in physical and chemical concentrations, including nutrients, are observed in river ecosystems. Understanding these cycles and separating the effects of physical loading (from point and non-point sources) and biogeochemical processes are necessary for water management and the TMDL process. A chloride mass balance approach is used to separate the relative influences of physical loading and biogeochemical processes in the Bow River through Calgary, Canada, which has a significant influence on the river water chemistry. Sampling campaigns were conducted in December 2005, when minimal photosynthesis and respiration occur, and in July 2006, when river discharge is high and some photosynthesis and respiration activity is present. Samples in each campaign were collected at point source input and output along the river reach through the city every hour for a 24-hour period, allowing for time of travel. The two wastewater treatment facilities within the city contributed the majority of physical mass loading to the river, with temporal variations in effluent discharge, chloride, and nutrient concentrations. Wastewater effluent chloride to nutrient (as well as other parameter relationships) concentrations also varied diurnally. An hourly chloride mass balance was achieved, within 0.5% (average, S.D.=4.4) for December and 7.7% (average, S.D.=4.2) for July, between estimated cumulative sum values from all inputs and measured values at the river output downgradient of the city, allowing for the investigation of other parameter conservativeness. Some slight diurnal variations associated with photosynthesis and respiration were observed even with limited productivity in the river. Nitrate mass fluxes appeared to be most strongly influenced by photosynthesis and respiration processes, with phosphate being less influenced. Ammonia mass fluxes appeared to be most strongly influenced by wastewater effluent loading. Physical loading can mask or enhance biogeochemical diurnal fluctuations, creating errors in river process interpretations. Chloride was a useful tracer in the mass balance to distinguish between and assist in separating physical loading and biogeochemical processes in the river.

Abnormal development of glomerular endothelial and mesangial cells in mice with targeted disruption of the lama3 gene.

Mice with targeted disruption of the lama3 gene, which encodes the alpha3 chain of laminin-5 (alpha3beta3gamma2, 332), develop a blistering skin disease similar to junctional epidermolysis bullosa in humans. These animals also develop abnormalities in glomerulogenesis. In both wild-type and mutant animals (lama3(-/-)), podocytes secrete glomerular basement membrane and develop foot processes. Endothelial cells migrate into this scaffolding and secrete a layer of basement membrane that fuses with the one formed by the podocyte. In lama3(-/-) animals, glomerular maturation arrests at this stage. Endothelial cells do not attenuate, develop fenestrae, or form typical lumens, and mesangial cells (MCs) were not identified. LN alpha3 subunit (LAMA3) protein was identified in the basement membrane adjacent to glomerular endothelial cells (GEnCs) in normal rats and mice. In developing rat glomeruli, the LAMA3 subunit was first detectable in the early capillary loop stage, which corresponds to the stage at which maturation arrest was observed in the mutant mice. Lama3 mRNA and protein were identified in isolated rat and mouse glomeruli and cultured rat GEnCs, but not MC. These data document expression of LAMA3 in glomeruli and support a critical role for it in GEnC differentiation. Furthermore, LAMA3 chain expression and/or another product of endothelial cells are required for MC migration into the developing glomerulus.

Evolution of novel functions: cryptocyanin helps build new exoskeleton in Cancer magister.

Hemocyanin, the blue blood protein of many arthropods and molluscs, reversibly binds oxygen at its highly conserved copper-oxygen-binding sites and supplies tissues with oxygen. Cryptocyanin, closely related structurally and phylogenetically to arthropod hemocyanin, lacks several of the six critical copper-binding histidines, however, and has lost the ability to bind oxygen. Despite this loss of function, cryptocyanin continues to be synthesized, an indication that it has been exploited to carry out new functions. Here, we show that cryptocyanin is present in extremely high concentrations in the hemolymph of the crab during the premolt portion of the molt cycle. Both proteins are specifically expressed in the same type of cell in the hepatopancreas and secreted into the hemolymph, but cryptocyanin plays a major role in forming the new exoskeleton, while hemocyanin functions in oxygen transport. A cessation in cryptocyanin, but not hemocyanin, synthesis after eyestalk ablation supports our hypothesis that cryptocyanin is closely regulated by molting hormones. The contrasts between the two gene products illustrate how a gene duplication of a copper-oxygen protein and its subsequent mutation may work in concert with the evolution of new regulatory mechanisms, leading to the assumption of new functions.

Changes in ground water quality in an irrigated area of southern Alberta.

Few studies have documented spatial and temporal variations in ground water quality in areas with high densities of animal farming operations (AFOs), or the long-term effects on surface-water quality. Changes in ground water quality were characterized in an irrigated area with a high density of AFOs in southern Alberta, Canada to evaluate the effect on ground water quality of manure application to fields. Fifty-five piezometers in the oxidized zone were sampled once or twice annually from 1995 to 2001, and temporal changes were analyzed using mixed model analysis. Average NO3- -N increased significantly from 12.5 to 17.4 mg L(-1) and average Cl- increased significantly from 19.4 to 34.4 mg L(-1) in piezometers installed in an unconfined sand aquifer at locations receiving fertilizer and manure. Compared with these manured locations, nitrate and chloride concentrations were significantly lower in shallow aquifer water in areas of pasture or native range, and concentrations did not change significantly with time. Nitrate and chloride concentrations in shallow ground water in fine-textured manured locations did not change significantly. Ground water below about 6 m in till and fine lacustrine sediments contains 18O signatures indicative of recharge under preirrigation or glacially influenced conditions, suggesting this ground water has a low vulnerability to agricultural contamination. Evaluations suggest that shallow ground water discharge will cause NO3- -N and Cl- in the Oldman River to increase by factors of at least 4.3 and 1.3, respectively, with more significant effects in smaller streams and under low-flow conditions.

Bacterial water quality in the personal water bottles of elementary students.

BACKGROUND: Samples of drinking water were collected directly from the personal water bottles of students at an elementary school in Calgary, Alberta. METHODS: Total and fecal coliforms and heterotrophic bacteria were enumerated using membrane filtration and agar plate count methods respectively. RESULTS: The Canadian Drinking Water Quality Guidelines (CWQG) criterion was exceeded for total coliform in 13.3% of 75 samples. Fecal coliform and total heterotrophic criteria were exceeded in 8.9% (of 68 samples) and 64.4% (of 76 samples) respectively. FINDINGS: The use of personal water bottles for students in elementary classrooms is not recommended.

HPA axis response to a psychological stressor in generalised social phobia.

Social phobia may be associated with a dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis. In this study we determined HPA axis responsivity to a psychological stressor in patients with social phobia and compared them to healthy controls. Fifteen patients with DSM IV social phobia with a mean score of 77.7 on the Liebowitz Social Anxiety Scale and 15 age and sex matched controls underwent the stressor consisting of mental arithmetic and a short term memory test performed in front of an audience. Plasma levels of cortisol and corticotropin were measured at various intervals throughout the test. Although baseline measures of cortisol did not differ between patients (319.8+/-34.6 nmol/l) and controls (279.5+/-42.7 nmol/l)(t=0.7, df=28, P<0.5) nor did baseline corticotropin values (8.6+/-2.1 pg/ml vs 13.7+/-2.0 pg/ml respectively) (t=-1.8, df=28, P<0.08) this stressor resulted in a significantly greater delta max cortisol response (the difference between baseline values and the maximum increase during the stressor) in patients (167.1+/-23.7 nmol/l) than in controls (106.7+/-16 nmol/l) (t=2.1, df=28, P<0.04). There was no significant difference in delta max corticotropin between groups (patients 8.8+/-2.1 pg/ml vs controls 9.1+/-1.9 pg/ml) (t=-0.08, df=28, P<0.9). This preliminary study indicates that patients with social phobia appear to have a hyper-responsive adrenocortical response to psychological stress.

Contrasting nitrate adsorption in Andisols of two coffee plantations in Costa Rica.

Fertilizer use in coffee plantations is a suspected cause of rising ground water nitrate concentrations in the ground water-dependent Central Valley of Costa Rica. Nitrate adsorption was evaluated beneath two coffee (Coffea arabica L.) plantations in the Central Valley. Previous work at one site had identified unsaturated zone nitrate retardation relative to a tritium tracer. Differences in nitrate adsorption were assessed in cores to 4 m depth in Andisols at this and one other plantation using differences in KCl- and water-extractable nitrate as an index. Significant adsorption was confirmed at the site of the previous tracer test, but not at the second site. Anion exchange capacity, X-ray diffraction data, extractable Al and Si, and soil pH in NaF corroborated that differences in adsorption characteristics were related to subtle differences in clay mineralogy. Soils at the site with significant nitrate adsorption showed an Al-rich allophane clay content compared with a more weathered, Si-rich allophane and halloysite clay mineral content at the site with negligible adsorption. At the site with significant nitrate adsorption, nitrate occupied less than 10% of the total anion adsorption capacity, suggesting that adsorption may provide long-term potential for mitigation or delay of nitrate leaching. Evaluation of nitrate sorption potential of soil at local and landscape scales would be useful in development of nitrogen management practices to reduce nitrate leaching to ground water.

Role of the peripheral myelin protein 22 N-linked glycan in oligomer stability.

Peripheral myelin protein 22 (PMP22) is a 22-kDa glycoprotein containing a single N-linked carbohydrate moiety. This posttranslational modification is conserved in PMP22 across species and within members of the PMP22 gene family; however, the function of the oligosaccharide is not known. To study the role of the PMP22 carbohydrate, site-directed mutagenesis was used to alter the glycosylation consensus sequence and produce a glycosylation-deficient mutant protein. This modified PMP22 was expressed in primary Schwann cells (SCs), and the effect of the N-glycan on the turnover rate, oligomerization, and intracellular trafficking of PMP22 was determined. Our data show a slight decrease in turnover rate from a half-life of approximately 70 min for the wild-type (wt) protein to 100 min for the glycosylation mutant. Although the presence of glycosylation-deficient PMP22 oligomers could be detected in SCs, we observed a decrease in oligomer stability compared with the wt oligomers. Both wt and mutant proteins showed similar localization in the endoplasmic reticulum and Golgi compartments and were transported to the SC surface. These results suggest that the N-glycan of PMP22 facilitates, in part, the stability of the PMP22 oligomer; however, the implications of PMP22 oligomerization remain unknown.

Galanin-R1 and -R2 receptor mRNA expression during the development of rat brain suggests differential subtype involvement in synaptic transmission and plasticity.

The present study employed 35S-labelled oligonucleotides and in situ hybridization to examine the distribution in the developing rat brain of mRNA encoding two galanin receptor subtypes, i.e. Gal-R1 and Gal-R2. Gal-R1 and/or Gal-R2 mRNA was detected at embryonic day (E) 20 and from postnatal day (P) 0-70. Gal-R1 mRNA was highly expressed in olfactory regions, ventral hippocampal CA fields, dorsomedial thalamic areas and many hypothalamic nuclei at all ages studied. In adult brain, Gal-R2 mRNA was most abundant in the dentate gyrus, anterior and posterior hypothalamus, raphe and spinal trigeminal nuclei, and in the dorsal motor nucleus of the vagus. At P0-P7, Gal-R2 mRNA was more widely distributed and abundant than at other ages, with highest levels of expression detected throughout the neocortex and thalamus. Thus, Gal-R2 transcripts had a more restricted distribution than Gal-R1 and were differentially abundant at different ages, while the distribution and relative abundance of Gal-R1 mRNA did not alter substantially during postnatal development. In general, Gal-R1 and -R2 mRNAs were localized in regions previously shown to contain [125I]-galanin binding sites and galanin-positive terminals in adult brain. Galanin-immunostaining was assessed in postnatal brain to determine whether peptide innervation correlated with observed transient receptor expression, but was not particularly enriched in Gal-R2 mRNA-positive areas of P4 or P7 brain. These results, together with earlier findings [e.g. Burazin, T. C. D. & Gundlach, A. L. (1998) J. Neurochem., 71, 879-882], suggest that Gal-R1 receptors have a broad role in normal synaptic transmission, while Gal-R2 receptors, in addition to a similar role in particular pathways, may be involved in processes prominent during the establishment and maturation of synaptic connections in developing brain and during neural damage and repair in the mature nervous system.

Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion.

Adhesion of keratinocytes in a wound outgrowth to laminin 5 in the basement membrane via integrins alpha6beta4 and alpha3beta1 is distinct from adhesion to dermal collagen via alpha2beta1 or to fibronectin via alpha5beta1. Leading cells in the outgrowth are distinguished from following keratinocytes by deposition of laminin 5, failure to communicate via gap junctions and sensitivity to toxin B, an inhibitor of RhoGTPase. Laminin 5 deposited by leading keratinocytes onto dermal collagen dominates over dermal ligands and changes the cell signals required for adhesion from collagen-dependent to laminin-5-dependent. Thus, deposition of laminin 5 can instruct keratinocytes to switch from an activated phenotype to a quiescent and integrated epithelial phenotype.

PMP22 accumulation in aggresomes: implications for CMT1A pathology.

Peripheral myelin protein 22 (PMP22) is a 22-kDa glycoprotein mainly expressed by Schwann cells (SCs). Duplication or deletion of the PMP22 gene locus is associated with heritable peripheral neuropathies suggesting that the correct level of PMP22 protein is essential for SC functioning. Previously we reported that in SCs the majority (80%) of newly synthesized PMP22 is rapidly degraded, possibly due to inefficient folding. Here we show that inhibition of the proteasome pathway results in a marked accumulation of PMP22 in the perinuclear cytoplasm. Double immunolabeling with an anti-ubiquitin antibody and various organelle markers indicates that the accumulated PMP22 is found in unique intracellular inclusions, called aggresomes. Moreover, overexpression of PMP22 in SCs can induce perinuclear accumulation of the protein. Together, these studies suggest that the proteasome pathway is critical for the regulation of PMP22 protein levels and raise the possibility that aggresomes may be involved in the pathogenesis of PMP22-associated peripheral neuropathies.

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.