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Biomedical subjects

E B Lane

Publications and source records attributed to E B Lane.

At least 19 recordsLinked to original sources
PubMed1995

Epitope mapping of monoclonal antibodies to keratin 19 using keratin fragments, synthetic peptides and phage peptide libraries.

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.

Amino Acid Sequence↗
PubMed1995

Comprehensive epitope analysis of monoclonal anti-proenkephalin antibodies using phage display libraries and synthetic peptides: revelation of antibody fine specificities caused by somatic mutations in the variable region genes.

Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pIII were used to define and localize the epitopes of 15 monoclonal antibodies raised against human proenkephalin, a neuropeptide precursor. Eight monoclonal antibodies (PE14 to PE19, PE23 and PE25), which inhibit each other's binding to proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it was possible to define the putative antibody epitope 155DLLKELL161 on human proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D(X)E(X)(X)V(X)(X)R could be defined with help of a 20mer library. The corresponding minimum epitope sequence has been found to be 175GSDNEEEVSKR185. Antibody PE1, raised in a separate fusion, was able to select phage clones from a 12mer and 20mer library, revealing that the sequence 187GGFMRG192 is probably the antibody epitope. The assumed localization of the epitopes was confirmed by screening a set of overlapping synthetic peptides, covering the region of human proenkephalin thought to contain all antibody binding sites. It was found that antibodies, although recognizing the same epitope, gave different binding patterns with the selected phage clones. By analysing the VH chain sequences of these antibodies it could be shown that a varying number of somatic mutations is likely to be the reason for the observed differences in antibody fine specificity.

Amino Acid Sequence↗
PubMed1995

Intermediate filaments in disease.

Intermediate filaments are major structural proteins encoded by a large multigene family. Their tissue-specific expression makes them important in studies of development, differentiation and pathology. Most intermediate filaments are keratins; recent discoveries of keratin mutations in a range of genetic skin disorders have clarified their role as providing essential structural support for cells in different physical settings.

DNA↗
PubMed1995

Keratin 16 and keratin 17 mutations cause pachyonychia congenita.

Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC.

Amino Acid Sequence↗
PubMed1995

A mutation in the mucosal keratin K4 is associated with oral white sponge nevus.

White sponge nevus (WSN) is a benign autosomal dominant disorder which affects non-cornifying stratified squamous epithelia (MIM 193900) (ref. 1). Phenotypically it presents as white 'spongy' plaques (oral leukokeratoses), most commonly in the mouth but also reported in the esophagus and anogenital mucosa. Histologically, the plaques show evidence of hyperproliferation, acanthosis and tonofilament aggregation. These types of pathogenic changes are characteristic of many of the epidermal keratin disorders. Keratins are expressed in pairs by epithelial cells in a tissue and cell specific manner. The major differentiation specific keratins of the buccal mucosa, nasal, esophageal and anogenital epithelia are K4 and K13 (ref. 7). The tissue distribution and nature of the lesions in patients affected by WSN suggested that mutations in K4 and/or K13 might be responsible for this disorder. We have now confirmed this hypothesis and report here a three base-pair (bp) deletion in the helix initiation peptide of K4 in affected members from two families with this condition.

Amino Acid Sequence↗
PubMed1995

Ultrastructural changes resulting from keratin-9 gene mutations in two families with epidermolytic palmoplantar keratoderma.

Palmoplantar keratoderma of Voerner type (or epidermolytic palmoplantar keratoderma) is an autosomal dominant inherited disorder of keratinization with histologic features of epidermolytic hyperkeratosis. We studied members of two large unrelated kindreds with epidermolytic palmoplantar keratoderma, and biopsy specimens of lesional palmar skin from both families confirmed the histologic changes of epidermolytic hyperkeratosis. Whorls of abnormally aggregated keratin filaments were seen ultrastructurally to be associated with signs of cellular disintegration in spinous and granular cells. Direct sequencing of genomic DNA samples obtained from several members of each family established the substitution of a highly conserved arginine by tryptophan (R162W) in the 1A region of the alpha-helical rod domain of keratin 9. This arginine residue in a highly conserved region of keratins 1 and 10 is affected by disruptive missense point mutations in many patients with bullous ichthyosiform erythroderma. An equivalent position in the sole and palm restricted keratin 9 appears to be the mutation hot spot in epidermolytic palmoplantar keratoderma. To date, R162W is the most prevalent genetic defect reported in this genodermatosis.

Base Composition↗
PubMed1995

Influence of temperature on long-term keratin immunoreactivity for oral exfoliative cytology.

We previously showed that keratin profiles can be of value in the diagnosis of oral cancer when using exfoliative cytology. In the future, they may form part of a screening program for oral cancer. This study evaluated the influence of long-term storage on keratin expression. Smears were collected from the clinically normal buccal mucosa and dorsal tongue of 22 patients. Half were stored in a refrigerator (5 degrees C) and half in a freezer (-70 degrees C). A total of 528 smears were collected. A panel of three antikeratin antibodies (LP34, AE8 and 1C7) was used to identify the preservation of keratin expression (graded as absent, few cells positive or many cells positive). The results for smears from dorsal tongue indicated that many cells were impermeable by the antikeratin antibodies. However, a satisfactory level of keratin immunoreactivity was observed in smears from buccal mucosa stored at -70 degrees C for over one year. Results for storage at 5 degrees C for both sites were inadequate after one month. Thus, smears from nonkeratinized oral sites may be stored at -70 degrees C for at least one year without a profound loss of keratin immunoreactivity, thus allowing examination of archival material.

Cold Temperature↗
PubMed1994

A functional "knockout" of human keratin 14.

The importance of keratins and other intermediate filaments in the maintenance of tissue structure is emphasized by the discovery that many hereditary skin-blistering diseases are caused by mutations in keratin genes. Here, we describe a situation in which keratin 14 (K14) is missing altogether in the epidermis: A homozygous 2-nucleotide deletion in exon I of the K14 gene causes premature termination of the mRNA transcripts upstream from the start of the rod domain and results in a K14 null phenotype. In this individual no keratin intermediate filaments are visible in basal epidermal cells, although filaments are present in the upper layers of the epidermis. No compensating keratin expression is detected in vivo, and K14 mRNA is down-regulated. The individual, diagnosed as Köbner (generalized) EBS, suffers from severe widespread keratinocyte fragility and blistering at many body sites, but although the phenotype is severe, it is not lethal. This K14-/- phenotype confirms that only one K14 gene is expressed in human epidermis and provides an important model system for examining the interdependence of different keratin filament systems and their associated structures in the skin.

Amino Acid Sequence↗
PubMed1994

A monoclonal antibody epitope on keratin 8 identified using a phage peptide library.

A bacteriophage random hexapeptide library was used to define the epitope of a monoclonal anti-keratin antibody. Phage selected by the keratin 8-specific antibody LE41 displayed highly related sequences on their pIII coat protein. The consensus sequence S(X)LNP allowed the precise localization of an LE41 epitope (SLLSP) within the head domain (H1 subdomain) of human keratin 8, known to be important for correct filament polymerisation. By sequencing the immunizing antigen, keratin 8 from Potorous tridactylis, it was shown that the natural epitope of LE41 is the pentapeptide SLLNP, which confirmed predictions from the phage library results. An SLL(X)P motif is found in the H1 region of all type II keratins (keratins 1 to 8) in different species, but mutational analysis revealed that LE41 can only bind to keratin 8 when Asn (N) or Ser (S) is found in the (X) position. Thus the monoclonal antibody LE41 retains its specificity for keratin 8, dependent on a single amino acid residue, even though it recognizes an epitope within the highly conserved H1 subdomain of the head region. Six other monoclonal antibodies tested on the phage library failed to select motifs.

Amino Acid Sequence↗
PubMed1994

Keratin diseases.

The recent discovery that epidermal fragility syndromes can be caused by mutations in the genes for keratin intermediate filaments has been a turning point for research into these structural proteins. Clustering of pathogenic mutations implies differential structural sensitivity along the keratin molecule, and implications for filament function require a new look at culture assay systems, plus a reassessment of structural defects in epithelial and other tissues.

Amino Acid Sequence↗
PubMed1994

DNA and keratin analysis of oral exfoliative cytology in the detection of oral cancer.

Refinements in oral exfoliative cytology may make it a suitable screening technique for the early diagnosis of oral cancer. In this study DNA range profiles were combined with keratin expression in an attempt to improve the diagnostic accuracy of oral exfoliative cytology. Smears were taken from 33 biopsy-proven oral cancers and the contralateral normal site. For DNA range profiles the smears underwent Feulgen hydrolysis, with DNA distribution being assessed using the Vickers M85 microdensitometer. For keratin expression a panel of antikeratin antibodies were applied. The smears for keratin expression were then graded on a three-point scale. Abnormal DNA range profiles were observed in 23 of 33 smears taken from oral cancers and in two smears from normal oral mucosa (sensitivity 70%, specificity 90%, positive predictive value 90%). The simple epithelial keratins 8 and 19 were identified in the majority of oral cancer smears. The sensitivity of keratin 19 was greater (90%). However, keratin 8 was the most useful keratin marker associated with malignancy (sensitivity 62%, specificity 100%, positive predictive value 100%). The combination of simple keratin expression and DNA content improved the cancer detection rate beyond that obtainable with DNA range profile alone.

Adult↗
PubMed1994

Ichthyosis bullosa of Siemens--a disease involving keratin 2e.

Ichthyosis bullosa of Siemens (IBS) is a congenital bullous ichthyosis without erythroderma. In contrast to bullous congenital ichthyosiform erythroderma (BCIE), there is a relatively mild involvement of the skin and epidermolytic hyperkeratosis (EHK) is restricted to the upper suprabasal layers of the epidermis. Tonofilament aggregation was observed by EM in suprabasal cells from affected patients in the two families under study, indicative of a keratin abnormality. Keratin 2e is a differentiation specific type II keratin expressed suprabasally in the epidermis. Part of the K2e gene was amplified by polymerase chain reaction using genomic DNA from affected and unaffected individuals from two IBS families. Direct sequencing of polymerase chain reaction products revealed a point mutation in the highly conserved helix termination motif, producing the protein sequence change LLEGEE-LLEGKE. This mutation was found in all affected members of a five-generation kindred and also in a sporadic case in a second unrelated family. No mutation was seen in unaffected individuals. The mutation destroys a MnlI restriction site, which allowed exclusion of the mutation from a population of 50 unaffected unrelated individuals by restriction fragment analysis of K2e PCR products. This is the sixth keratin gene found to be involved in an inherited epidermal disorder.

Base Sequence↗
PubMed1994

Ichthyosis bullosa of Siemens is caused by mutations in the keratin 2e gene.

Ichthyosis bullosa of Siemens is a blistering disorder with autosomal dominant inheritance. The disease resembles bullous congenital ichthyosiform erythroderma but is less severe. Keratins K1 and K10 have been implicated in bullous congenital ichthyosiform erythroderma. Linkage analysis pointed to the involvement of a keratin type II gene (12q11-13) in ichthyosis bullosa of Siemens. Mutations in the highly conserved regions of K1, a member of the type II gene cluster, were excluded. The gene coding for keratin 2e is also located in the type II gene cluster and the expression of the gene coincides with the occurrence of epidermolytic hyperkeratosis. Sequence analysis revealed the presence of mutations in the K2e gene in patients with ichthyosis bullosa of Siemens. Three different mutations were detected, one in the 1A domain and two in the 2B domain of the rod. Furthermore, histologic and ultrastructural examination of skin biopsies indicated that ichthyosis exfoliativa is identical to ichthyosis bullosa of Siemens. This was confirmed by the results of the molecular analysis. In the family diagnosed as ichthyosis exfoliativa, a mutation was detected that was identical to the mutation found in one of the families with ichthyosis bullosa of Siemens.

Adult↗
PubMed1994

Keratinocyte differentiation in psoriatic scalp: morphology and expression of epithelial keratins.

The morphology of hair follicles was examined in psoriatic scalp biopsies and compared with normal scalp. In scalp psoriasis the lower outer root sheath and hair matrix were not affected by the psoriatic changes, although there was an irregular expansion in the proximal lower outer root sheath. This area has been characterized, by the presence of keratin K19-containing cells, as the putative stem cell region. In addition, marked shrinkage of the sebaceous glands was seen in the psoriatic scalp, as previously reported. A panel of monospecific monoclonal antibodies to individual epithelial keratins was used to analyse scalp specimens immunohistochemically. Keratin expression in scalp was generally unaffected by psoriasis, except for widespread expression of suprabasal keratins K16 and K17 in suprabasal interfollicular psoriatic scalp epidermis. Simple epithelial keratins K8 and K18 were not found in follicular epithelium from either normal or psoriatic scalp, using multiple monospecific antibodies. This study shows that keratin K17 is induced suprabasally during epidermal hyperproliferation, and cannot therefore be considered a hair follicle-specific keratin.

Adolescent↗
PubMed1994

Protein and mRNA expression of simple epithelial keratins in normal, dysplastic, and malignant oral epithelia.

Simple epithelial keratins K7, K8, and K18 are present in no more than trace amounts in normal stratified squamous epithelial but have been reported in squamous cell carcinomas. With the aim of determining the level at which keratin synthesis is regulated in vivo, we have compared the expression of mRNA by in situ hybridization and protein by immunohistochemistry for K7, K8, and K18 in a series of normal, dysplastic, and malignant oral epithelia. In normal epithelia mRNAs for K7, K8, and K18 were present in basal and lower spinous cells but adjacent sections were generally negative for the respective proteins. In severe dysplasia there was irregular suprabasal extension of K8 and K18 mRNAs in all cases and their proteins were expressed in more than half of the cases. The carcinomas expressed K8 and K18 mRNAs homogeneously and were strongly reactive for these keratin proteins but K7 expression appeared reduced in malignancy. These results are consistent with the post-transcriptional regulation of K7, K8, and K18 expression in normal epithelia and the presence of their proteins in dysplastic and malignant epithelia suggests the release of these epithelial cells from a post-transcriptional block on K8 and K18 translation. Alternatively, rapid degradation of K8 and K18 protein might be occurring in normal epithelia but be suppressed in dysplasia and malignancy.

Base Sequence↗
PubMed1993

Expression of keratin mRNAs and proteins in normal salivary epithelia and pleomorphic adenomas.

Control of keratin (K) gene expression may be important for cell differentiation in complex epithelia such as salivary gland. To investigate differences in distribution between keratin mRNAs and their respective proteins, a combined in situ hybridization (ISH) and immunohistochemical study was undertaken on nine normal salivary glands and seven pleomorphic adenomas. ISH employed riboprobes to K7, K8, K14, K18, and K19. Immunohistochemistry was performed on adjacent sections using monoclonal antibodies (MAbs) to the same keratins. Normal luminal cells showed abundant hybridization with probes for K7, K8, K18, and K19. Keratin 14 mRNA was present in basal and myoepithelial cells at a low level of expression. Proteins of their keratins were strongly stained. Neoplastic cells showed variable expression of mRNA and protein for K7, K8, K18, and K19. There was a high level of K14 mRNA but variable protein. The findings provide evidence that expression of these keratins in normal salivary epithelia is regulated transcriptionally and that in neoplasia this system is in considerable disarray.

Adenoma, Pleomorphic↗
PubMed1993

Network incorporation of intermediate filament molecules differs between preexisting and newly assembling filaments.

When studying the way in which intermediate filaments assemble in vivo, it is important to distinguish between the incorporation of intermediate filament proteins into an existing intermediate filament network and the ability to form a new network within cells. To distinguish between these alternatives, we have made a hybrid construct consisting of the rod and tail domains of murine glial fibrillary acidic protein (GFAP) coupled to the head domain of bovine keratin 19, called K19GFAP. The assembly characteristics of K19GFAP were analyzed in vitro and in vivo. Replacement of the head domain with the bovine K19 sequence did not prevent the incorporation of K19GFAP into the existing network of vimentin intermediate filaments in NIH 3T3 cells but it was incompatible with de novo formation of filament networks in the epithelial cell line MCF-7, which lacks an endogenous vimentin network. By in vitro assembly studies, it was confirmed that K19GFAP was unable to assemble into typical intermediate filaments. We also investigated the ability of an appropriate type II keratin partner to rescue K19GFAP from incorporation into a vimentin network and initiate de novo filament assembly, using the fibroblast cell line KF-K8(3), an NIH 3T3 fibroblast cell line expressing a single human keratin, K8. The results confirm the importance of the coiled coil interactions in determining the fate of intermediate filament proteins. The results also emphasize that filament networks can not only tolerate but also incorporate assembly-deficient intermediate filament protein subunits.

3T3 Cells↗