High affinity of alpha-foetoprotein for arachidonate and other fatty acids.

Fatty acid analysis of purified bovine alpha-foetoprotein showed it to contain 2.7 mol of fatty acid/mol of alpha-foetoprotein. Purified alpha-foetoprotein focused at isoelectric point 4.8. Removal of bound ligands from alpha-foetoprotein by charcoal treatment changed its isoelectric point to 5.2. This change could be reversed by addition of exogenous fatty acids to the defatted alpha-foetoprotein. Albumin isolated from the same foetal calf serum source as alpha-foetoprotein contained 1.4 mol of fatty acid/mol of protein. alpha-Foetoprotein and albumin contained comparable amounts of fatty acids with 14 to 18 carbon atoms, but alpha-foetoprotein contained 16 times as much of the long-chain polyunsaturated fatty acids as albumin. alpha-Foetoprotein was found to have slightly higher affinity for palmitate and linoleate and severalfold higher affinity for arachidonate than albumin. These findings suggest that alpha-foetoprotein may play a role in the foetal metabolism of the long-chain polyunsaturated fatty acids.     

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.     

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.     

Identification, expression, and localization of β keratin gene products during development of avian scutate scales

Epidermal-dermal interactions influence morphogenesis and expression of the beta keratin gene family during development of scales in the embryonic chick. The underlying mechanisms by which these interactions control beta keratin expression are not understood. However, the present study of beta keratin gene expression during avian epidermal differentiation contributes new information with which to investigate the role of tissue interactions in this process. Using beta keratin-specific synthetic oligonucleotide probe, beta keratin mRNA was hybrid-selected from total poly A+ RNA of scutate scales. Seven beta keratin polypeptides were translated in vitro and could be identified by their positions in two-dimensional gels among the detergent-insoluble extracts of scutate scale epidermis. In vivo phosphorylation studies suggested that an additional three beta keratin polypeptides were present as phosphoproteins. The temporal appearance of beta keratin mRNA and the corresponding polypeptides was followed during scutate scale development. Polyclonal antiserum made against two of the beta keratin polypeptides was used for immunohistochemical and immunogold electron-microscopic analysis of beta keratin tissue distribution. Immunological reactivity was observed specifically along the outer scale surface in epidermal cells above the stratum germinativum. Immunogold beads were localized on 3-nm filament bundles. In situ hybridization with a beta keratin-specific RNA probe demonstrated that mRNA accumulated in the same regional manner as the polypeptides. This selective expression of beta keratin genes in specific regions of the developing scutate scale suggests that epidermal-dermal interactions provide not only for morphological events, but also for control of complex patterns of histogenesis and biochemical differentiation.     

Role of dehydration in changing the permeability of erythrocyte plasma membranes by freeze-thawing

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing 32Pi, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weights between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [gamma-32P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [gamma-32P]ATP and cyclic AMP-dependent protein kinase from calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives

Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.     

A role for phosphorylation in the dynamics of keratin intermediate filaments

Keratins undergo highly dynamic events in the epithelial cells that express them. These dynamic changes have been associated with important cell processes. We have studied the possible role of keratin phosphorylation-dephosphorylation processes in the control of these dynamic events. Drugs that affect the protein phosphorylation metabolism (activators or inhibitors of protein kinases or protein phosphatases) have been used in two different dynamic experimental systems. First, the behaviour of keratins after the formation of cell heterokaryons, and second, the assembly of a newly synthesised keratin after transfection into the pre-existing keratin cytoskeleton. The main difference between these two systems stems on the alteration of the amount of keratin polypeptides present in the cells, since in heterokaryons this amount was unaltered whilst in transfection experiments there is an increase due to the presence of the transfected protein. We observed in both systems that the inhibition of protein kinases led to a delayed dynamic behaviour of the keratin polypeptides. On the contrary, the inhibition of protein phosphatases by okadaic acid or the activation of protein kinases by phorbol esters promoted a substantial increase in the kinetics of these processes. Biochemical studies demonstrate that this behavioural changes can be correlated with changes in the phosphorylation state of the keratin polypeptides. As a whole, present results indicate that the highly dynamic properties of the keratin polypeptides can be modulated by phosphorylation.     

Phosphorylation of keratin polypeptides

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing ³²P_i, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weight between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [γ-³²P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [γ-³²P]ATP and cyclic AMP-dependent protein kinase form calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Keratin Polypeptide Analysis in Fetal and in Terminally Differentiating Newborn Mouse Epidermis

The keratin polypeptide pattern of neonatal mouse epidermis consists of eight individual polypeptides having molecular weights of between 46,000 and 67,000. Unlike the keratin patterns in adult mouse epidermis, this pattern is not subject to body site-specific alterations regarding the specific content of distinct polypeptides or the absolute number of keratin constituents.
At day 16 of fetal development the neonatal keratin pattern is only partially expressed, it being fully completed just prior to birth at day 19 of gestation. A comparative analysis of the sequential changes in epidermal morphology and keratin pattern during the last third of embryonic development confirms the view that, independent of the species, keratin polypeptides below 60,000 mol. wt. are generated by basal cells, whereas the biosynthesis of high molecular weight keratin members take place in the suprabasal cell compartments of keratinizing epithelia. The site of origin of five polypeptides (60,000, 58,000, 52,000, 49,000, 46,000) could therefore be attributed to the basal cell layer, the remaining three polypeptides (67,000, 64,000, 62,000) being synthesized in the outer metabolically active epidermal layers. Similar conclusions could be drawn after subfractionation of neonatal epidermis into living (basal, spinous, and granular) and dead cell layers (stratum corneum), and investigation of the corresponding keratin patterns.
During their progression through the epidermis, two proteins (60,000, 58,000) undergo a hitherto undescribed type of posttranslational modification characterized by a slight increase in size and a change in electrical charge. The mechanism underlying this modification is unknown and it is unclear whether the modification if functional or trivial. The largest keratin polypeptide (67,000) of the protein family - probably a product of spinous cells - disappears from the cornified layer without any evidence that it serves as a precursor for smaller keratin subunits.     

Keratin polypeptide expression in mouse epidermis and cultured epidermal cells

Adult mouse epidermis contains up to 11 distinct keratin polypeptides, as resolved by two-dimensional gel electrophoresis. These include both basic (Type II; 67-, 65-, 63-, 62-, and 60-kDa) and acidic (Type I; 61- to 59-, 54-, 52-, 49-, and 48-kDa) keratins that exhibit multiple isoelectric forms. Several, but not all, of these keratins, identified by immunoblotting, were found to be actively synthesized in the skin when assayed in short-term pulse-labeling experiments. When compared to the adult, newborn mouse epidermis expresses fewer keratin subunits. However, greater amounts of keratins associated with differentiated suprabasal cells and stratum corneum, which is more pronounced morphologically in the newborn, were identified. We also observed strain-specific differences in the expression of a Type I acidic keratin. This 61-kDa (pI, approx. 5.3) keratin was produced exclusively by the CF-1 mouse and, based on peptide mapping, appeared to be related to the acidic 59-kDa keratin that was identified in this strain as well as all other mouse strains. The 61-kDa keratin was not expressed in vitamin A-deficient animals, suggesting that its appearance may be related to a retinoid-dependent posttranslational modification. In comparison to keratin expression in vivo, primary mouse keratinocyte monolayer cultures maintained in low Ca2+ (less than 0.08 mM) did not express the terminal differentiation keratins of 67-kDa (basic) or 59-kDa (acidic), although enhanced synthesis of the 60-kDa (basic) and the 52-kDa and 59-kDa (acidic) keratins associated with proliferation were observed. In addition, a subpopulation of nonadherent cells was continuously produced by the primary keratinocyte cultures that expressed the 67-kDa (basic) keratin specific for terminal differentiation. When the keratinocyte cultures were induced to terminally differentiate with Ca2+, the overall pattern of keratin expression was not changed significantly. Taken together, these results provide further evidence for the variable nature of keratin expression in mouse epidermal keratinocytes under different growth conditions.     

Keratin polypeptide analysis in fetal and in terminally differentiating newborn mouse epidermis

The keratin polypeptide pattern of neonatal mouse epidermis consists of eight individual polypeptides having molecular weights of between 46,000 and 67,000. Unlike the keratin patterns in adult mouse epidermis, this pattern is not subjects to body site-specific alterations regarding the specific content of distinct polypeptides or the absolute number of keratin constituents. At day 16 of fetal development the neonatal keratin pattern is only partially expressed, it being fully completed just prior to birth at day 19 of gestation. A comparative analysis of the sequential changes in epidermal morphology and keratin pattern during the last third of embryonic development confirms the view that, independent of the species, keratin polypeptides below 60,000 mol. wt. are generated by basal cells, whereas the biosynthesis of high molecular weight keratin members take place in the suprabasal cell compartments of keratinizing epithelia. The site of origin of five polypeptides (60,000, 58,000, 52,000, 49,000, 46,000) could therefore be attributed to the basal cell layer, the remaining three polypeptides (67,000, 64,000, 62,000) being synthesized in the outer metabolically active epidermal layers. Similar conclusions could be drawn after subfractionation of neonatal epidermis into living (basal, spinous, and granular) and dead cell layers (stratum corneum), and investigation of the corresponding keratin patterns. During their progression through the epidermis, two proteins (60,000, 58,000) undergo a hitherto undescribed type of posttranslational modification characterized by a slight increase in size and a change in electrical charge. The mechanism underlying this modification is unknown and it is unclear whether the modification if functional or trivial. The largest keratin polypeptide (67,000) of the protein family -- probably a product of spinous cells -- disappears from the cornified layer without any evidence that it serves as a precursor for smaller keratin subunits.     

Characterization of the keratin filament subunits unique to bovine snout epidermis.

Steinert [Biochem. J. (1975) 149, 39-48] reported that the alpha-keratin polypeptides (the subunits of the intracellular keratin filaments) of bovine hoof and snout epidermis are the same. We now demonstrate that this is not so: in addition to the seven polypeptides previously identified in hoof epidermis, snout epidermis also contains at least three other polypeptides of higher molecular weight. These unique polypeptides were isolated, purified and characterized. They are chemically and structurally very similar to the other polypeptides of bovine epidermis and readily polymerize in vitro with them to form native-type epidermal keratin filaments.     

Proteomics of a new esophageal cancer cell line established from Persian patient

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study.     

Effects of retinoic acid and other retinoids on the growth and differentiation of 3T3 supported human keratinocytes

Using mitomycin C treated 3T3-Swiss fibroblasts as feeder cells, human keratinocytes derived from infant foreskins were subcultured in the presence of trans-retinoic acid (RA) and other retinoids. At 1 microgram/ml (3 x 10(-6) M) and higher RA concentrations plating efficiency was markedly reduced. Addition of retinoids to 1 microgram/ml after colonies were established produced no change in the rate of cell production, but caused differentiated cells to be sloughed earlier than in control cultures. Electron microscopy showed wider extra cellular spaces the contained numerous villi, increased numbers of microvilli at the surfaces of the outermost cells, and decreased number of cell layers all of which were consistent with the observed desquamatory effects of RA. Retinoic acid also extended the time during which cell population increased so that RA treated cultures produced more cells than control cultures over their respective lifetimes. Keratin polypeptides represented a smaller percentage of the total solubilizable protein and more keratin was present as acid soluble prekeratin in postconfluent RA treated cultures. This may be a consequence of early desquamation rather than a decrease in keratin synthesis. No unusual proteins were visible in RA treated cultures by simple sodium dodecylsulfate electrophoresis, nor was there increase in specific activities of three lysosomal enzymes.     

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

Chemical reception mechanisms at a low level of phylogeny. Influence of polypeptide hormones and non-hormone polypeptides on the growth of Tetrahymena

The polypeptide hormones insulin, glucagon, thyrotropin (TSH), pregnant mare serum gonadotropin (PMSG) and adrenocorticotropin (ACTH) stimulated the growth of the Tetrahymena, and the non-hormone polypeptides (bovine serum albumin (BSA), protamine) had a similar effect. Re-exposure after 24 h accounted for a greater growth stimulation than pre-exposure alone in cultures treated with TSH and PMSG, and re-exposure after 7 days had such effect in all polypeptide-treated cultures. It follows that the non-hormone polypeptides had a similar imprinting potential to the polypeptide hormone. The non-hormone polypeptides were also able to cross-imprint for one another, i.e. pre-exposure to one enhanced the binding capacity of the cells for the other on re-exposure, and vice versa. A single treatment with a polypeptide hormone or a non-hormone polypeptide did in itself stimulate the growth of the Tetrahymena for as long as 1 week.     

Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant

The two major intermediate filament proteins in glandular epithelia are keratin polypeptides 8 and 18 (K8/18). To evaluate the function and potential disease association of K18, we examined the effects of mutating a highly conserved arginine (arg89) of K18. Expression of K18 arg89-->his/cys and its normal K8 partner in cultured cells resulted in punctate staining as compared with the typical filaments obtained after expression of wild-type K8/18. Generation of transgenic mice expressing human K18 arg89-->cys resulted in marked disruption of liver and pancreas keratin filament networks. The most prominent histologic abnormalities were liver inflammation and necrosis that appeared at a young age in association with hepatocyte fragility and serum transaminase elevation. These effects were caused by the mutation since transgenic mice expressing wild-type human K18 showed a normal phenotype. A relative increase in the phosphorylation and glycosylation of detergent solubilized K8/18 was also noted in vitro and in transgenic animals that express mutant K18. Our results indicate that the highly conserved arg plays an important role in glandular keratin organization and tissue fragility as already described for epidermal keratins. Phosphorylation and glycosylation alterations in the arg mutant keratins may account for some of the potential changes in the cellular function of these proteins. Mice expressing mutant K18 provide a novel animal model for human chronic hepatitis, and for studying the tissue specific function(s) of K8/18.