The hSkn-1a POU transcription factor enhances epidermal stratification by promoting keratinocyte proliferation.

Skn-1a is a POU transcription factor that is primarily expressed in the epidermis and is known to modulate the expression of several genes associated with keratinocyte differentiation. However, the formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation, and a role for Skn-1a in this process has not been previously demonstrated. Here, our results show, surprisingly, that human Skn-1a contributes to epidermal stratification by primarily promoting keratinocyte proliferation and secondarily by enhancing the subsequent keratinocyte differentiation. In organotypic raft cultures of both primary human keratinocytes and immortalized HaCaT keratinocytes, human Skn-1a expression is associated with increased keratinocyte proliferation and re-epithelialization of the dermal substrates, resulting in increased numbers of keratinocytes available for the differentiation process. In these same raft cultures, human Skn-1a expression enhances the phenotypic changes of keratinocyte differentiation and the upregulated expression of keratinocyte differentiation genes. Conversely, expression of a dominant negative human Skn-1a transcription factor lacking the C-terminal transactivation domain blocks keratinocytes from proliferating and stratifying. Keratinocyte stratification is dependent on a precise balance between keratinocyte proliferation and differentiation, and our results suggest that human Skn-1a has an important role in maintaining epidermal homeostasis by promoting keratinocyte proliferation.

Characterization of the regulatory domains of the human skn-1a/Epoc-1/Oct-11 POU transcription factor.

The Skn-1a POU transcription factor is primarily expressed in keratinocytes of murine embryonic and adult epidermis. Although some POU factors expressed in a tissue-specific manner are important for normal differentiation, the biological function of Skn-1a remains unknown. Previous in vitro studies indicate that Skn-1a has the ability to transactivate markers of keratinocyte differentiation. In this study, we have characterized Skn-1a's transactivation domain(s) and engineered a dominant negative protein that lacked this transactivation domain. Deletional analysis of the human homologue of Skn-1a with three target promoters revealed the presence of two functional domains: a primary C-terminal transactivation domain and a combined N-terminal inhibitory domain and transactivation domain. Skn-1a lacking the C-terminal region completely lost transactivation ability, irrespective of the promoter tested, and was able to block transactivation by normal Skn-1a in competition assays. Compared with full-length, Skn-1a lacking the N-terminal region demonstrated either increased transactivation (bovine cytokeratin 6 promoter), comparable transactivation (human papillomavirus type 1a long control region), or loss of transactivation (human papillomavirus type 18 long control region). The identification of a primary C-terminal transactivation domain enabled us to generate a dominant negative Skn-1a factor, which will be useful in the quest for a better understanding of this keratinocyte-specific gene regulator.

alpha1-antitrypsin gene mutation hot spot associated with the formation of a retained and degraded null variant [corrected; erratum to be published].

Null alpha1-antitrypsin (alpha1AT) alleles represent the end of a continuum of variants associated with profound alpha1AT deficiency and an increased risk of emphysema. This study characterizes the molecular basis of QOclayton, a new example of an alpha1AT null allele arising from a mutational hot spot in the alpha1AT gene. The QOclayton allele is identical to the normal M1(V213) alpha1AT allele except for an insertion of a cytosine. This insertion occurs in the alpha1AT sequence which normally has seven cytosines corresponding to amino acid residues 360 to 362. The QOclayton mutation is located in the same reiterated DNA sequence as the alpha1AT QObolton deletion mutation and the insertion mutation allele QOsaarbruecken. The QOclayton cytosine insertion causes a 3' frameshift and results in the formation of a termination codon at residue 376, the same consequence as the alpha1AT QOmattawa mutation (L353 T-insertion with a 3' frameshift). To determine the molecular mechanisms responsible for the absence of alpha1AT associated with the QOclayton gene, an in vitro model of QOclayton was established using Chinese hamster ovary cells (CHO) transfected with the QOclayton gene. These cells were evaluated for alpha1AT mRNA expression, protein synthesis and secretion. Although the QOclayton gene expresses a similar amount of alpha1AT mRNA as compared with the normal alpha1AT gene, no QOclayton protein is secreted. Protein trafficking and double-label immunofluorescence demonstrate that the QOclayton protein is retained in the rough endoplasmic reticulum or pre-Golgi compartment and is degraded (t1/2 = 6.5 h). Since QOmattawa, QObolton, and QOsaarbruecken have similar termination sites in the alpha1AT mRNA, they may share a similar intracellular fate.

Genetic diversity from a limited repertoire of mutations on different common allelic backgrounds: alpha 1-antitrypsin deficiency variant Pduarte.

alpha 1-Antitrypsin (alpha 1AT) is one of the most polymorphic gene loci in the human genome. alpha 1AT variants are typically identified by their migration position in an isoelectric focusing gel at pH 4-5. Heterogeneity of the isoelectric point of alpha 1AT variants, hence variant migration, most often results from amino acid substitutions which alter the net charge of the molecule. We identified an individual heterozygous for an alpha 1AT variant migrating in the "P" variant region which differs from other known "P" variants. Using isoelectric focusing on an immobilized pH gradient at pH 4.50-4.85 the novel P allele, Pduarte, migrates between Pst. albans and Plowell. Densitometric analysis of normal "M" type alpha 1AT and the deficiency variant Plowell major bands separated by isoelectric focusing demonstrates that Pduarte contributes approximately 41% as much alpha 1AT to the total serum alpha 1AT concentration as the normal "M" alpha 1AT, similar to Plowell. Direct DNA sequencing of the proband's genomic DNA demonstrates that the Pduarte allele differs from the normal M1 (V213) allele by two amino acid substitutions, R101 (CGT)-->H(CAT) and D256 (GAT)-->V(GTT). Individually, these amino acid substitutions characterize the normal M4 allele (R101-->H) and the deficient Plowell allele (D256-->V). Thus the Pduarte allele differs from the Plowell allele only by the normal allelic background in which the V256 mutation occurs. Comparison of amino acid sequences among several alpha 1AT variants demonstrates that Pduarte is an example of a more general observation regarding diversity within the PI (protease inhibitor) system.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of corneal epithelial wound healing rates in scrape vs. lamellar keratectomy injury.

Previous in vivo studies evaluating the effects of growth factors on epithelial regeneration have used the scrape injury model in rabbit eyes. Since growth factors act principally on the mitotic activity of regenerating cells, the rapid wound closure rates following scrape injury may not adequately access the effects of these agents on epithelial repair. In this study, we evaluated the rates of wound healing following scrape (8.6 mm) and lamellar keratectomy (8.6 mm) injury in 25 albino rabbits. Eyes were left untreated or received daily application (two to three times) of (a) Tears Naturale II, (b) 50 mM Tris/NaCl, and (c) commercial vehicle for EGF. Eyes were evaluated daily by fluorescein staining with Ophthalmic Fluoro-Strips followed by clinical photography. The area of staining was quantitated by computer-assisted planimetry and rates calculated by linear regression analysis. Eyes receiving scrape injuries epithelialized by 3 days following surgery. Rates of wound closure in two separate groups (six eyes each) were 25.83 mm2/day (r = 0.96) and 29.56 mm2/day 9r = 0.97). Lamellar keratectomy injuries epithelialized by 7 to 8 days, which is substantially longer than that observed for scrape injuries. Rates of wound healing in two separate untreated groups (8 and 10 eyes) were 10.88 mm2/day (r = 0.95) and 9.00 mm2/day (r = 0.93), respectively, which were not significantly different. Analysis of variance comparing rates of wound closure indicated that lamellar keratectomy injuries heal at a significantly slower rate when compared to scrape injury (p less than 0.0005).(ABSTRACT TRUNCATED AT 250 WORDS)

First isolation step of a plasma factor which enhances lymphocytic cortisol metabolism and which is reduced in cancer patients.

Our previous findings show that cancer patients' plasma lacks a factor (LCMEF) which is characterized by its ability to enhance lymphocytic cortisol metabolism. In the present study, using the dialysis method, we tried to evaluate the size of that factor. Plasma of healthy donors (HP) was dialyzed against water. Human lymphocytes from healthy donors were incubated with cortisol in media containing buffer (PBS) and one of the following additions: 1) dialyzate, 2) dialyzed HP (DP), 3) reconstituted HP (DP + P). The cortisol conversion rates were compared with that obtained with untreated HP and with PBS. The results showed no significant differences among the conversion rates obtained with HP, dialyzate, and reconstituted HP. Dialyzed plasma, however, showed a significantly lower rate, which was equal to that obtained with PBS. This implies that the factors are smaller than 10,000 daltons.