Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12.

Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.

Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K12 keratin is restricted to corneal epithelium.

The conjunctival epithelium is intrinsically different from the corneal epithelium in vivo, but sometimes can transform into an epithelium morphologically indistinguishable from the latter after healing of a total corneal epithelial defect. It remains unclear whether this morphologic transformation represents a process of extrinsic modultation or transdifferentiation of intrinsically divergent epithelium. In air-lifted organotypic cultures, rabbit conjunctival epithelial cells lost goblet cell differentiation and were stratified to the same extent as corneal epithelial cells, resembling the above in vivo morphologic transformation. Paired expression of K3 (64 kD) and K12 (55 kD) keratins has been regarded as a marker for corneal-type differentiation. Immunoblot analysis by monoclonal antibody AE5 revealed that K3 keratin was expressed by both submerged or air-lifted corneal and conjunctival cultures with or without 3T3 fibroblasts in collagen gel. In contrast, K12 keratin was expressed only by air-lifted corneal cultures with 3T3 fibroblasts using monoclonal antibody AK2 and two epitope-specific antibodies to N- and C- terminal oligopeptides deduced from the mouse K12 gene. This finding was also confirmed by Northern hybridization with a rabbit K12 cDNA probe. The expression of K12 keratin was more delayed than that of K3 keratin in air-lifted corneal cultures. This dissociated expression of these two keratins resembles that noted in vivo in the stem cell-containing limbal region. These results suggest that morphologic transformation of the conjunctival epithelium represents extrinsic environment modulation, and that differential expression of K12 but not K3 keratin signifies corneal epithelial differentiation.

Improved allograft survival using highly enriched populations of rat islets.

A method is described for the purification of islets before the cells are placed in tissue culture, thus permitting the transplantation of islets cultured for three days against major histocompatibility barriers without adjuvant immunosuppression. Mixed lymphocyte culture reactions were carried out with three rat strain combinations and the in vitro responses were correlated with the in vivo survival of islet allografts. These results showed that islet allograft acceptance is independent of the degree of histoincompatibility between different rat strains.

The effect of donor strain blood and ALS therapy on pancreatic islet allograft survival in the rat.

Small amounts of donor strain ACI blood injected in conjunction with minimal ALS therapy prolonged islet allograft survival in Wistar-Lewis, diabetic recipients from a mean of 8 days in nontransfused rats to a mean of more than 100 days. ACI skin grafts transplanted on long-surviving transplant-improved rats were rejected within 8 days, which suggested that "classical" tolerance was probably not the mechanism responsible for the blood-induced suppression of islet allograft rejection.

Cyclic AMP and psoriasis.

Evidence that an adenyl cyclase system is present in all mammalian epidermis is reviewed. This adenyl cyclase is stimulated by at least two separate types of chemicals: catecholamines, which act at a beta-adrenergic receptor site, and prostaglandins of the E series, which act at a separate site. In the psoriatic lesion, the response to these stimulators, especially to the catecholamines, is reduced. Despite this lack of response to external agents which elevate cyclic AMP, the concentration of cyclic AMP within the epidermis of the psoriatic lesion is no lower than in noninvolved skin. How cyclic nucleotides act to control cell proliferation and cell differentiation remains unclear.

Comparison of 12alpha-hydroxylation of oxygenated 5alpha-cholestanes and allochenodeoxycholate with rat liver microsomes.

[5alpha,6alpha-3H2]-5alpha-Cholestane-3alpha,7alpha-diol (A), (25 R)-3alpha,7alpha-dihydroxyl-[5alpha,6alpha-3H2]-5alpha--cholestan-26-oic acid (B),(25R)-[5alpha,6alpha-3H2]stane-3alpha,7alpha-26-triol (C), and [3beta-3H]allochenodeoxycholic acid (D) were prepared, characterized, and studied with a rat liver microsomal preparation fortified with 1 mM NADPH. The 12alpha-hydroxylated product formed from each of these substrates was identified by isotopic dilution; the relative reactivity of the four substrates was (A) 100; (B) 87; (C) 135; and (D) 40. The microsomal system showed a requirement for added NADPH and other properties similar to those shown for 12alpha-hydroxylation of 7alpha-hydroxycholest-4-en-3-one, which is ultimately converted to cholic acid. Since the coplanar 5alpha-cholestane-3alpha,7alpha-diol is virtually superimposable upon 7alpha-hydroxycholest-4-en-3-one, and the enzymic requirements are comparable, it is suggested that a single enzyme system may be responsible for 12alpha-hydroxylation of these substrates.

XLV. Metabolism of 3beta,7alpha-dihydroxy-5alpha-cholestanoic acid in the rat with a bile fistula.

[25R]3beta,7alpha-Dihydroxy-[5alpha,6alpha- -3 H2]-5alpha-cholestanoic acid was prepared, purified and 0.34 mg was administered intraperitoneally as the potassium salt to each of three adult male rats with cannulated bile ducts. Bile collected in the first 24 h, containing 97% of the administered 3-H was hydrolysed and the free bile acids were separated by acetic acid partition chromatography. Of the chromatographed tritium 58% was associated with allochenodeoxycholic acid and 14% with its 3beta-isomer; only 5% of the 3-H was found in allocholic acid and 1% with the substrate or more polar unidentified materials. Thus, this dihydroxy-5alpha-cholestanoic acid is metabolized in the rat primarily to dihydroxy-5alpha-cholanic acids, comparable to the metabolism of 3alpha, 7alpha-dihydroxy-5beta-cholestanoic acid in man.

Further studies on adenyl cyclase in psoriasis.

Slices of human skin obtained with a keratome were pre-incubated with [3H]adenine to label the ATP pool from which cyclic AMP was subsequently formed. The accumulation of radioactive cyclic AMP was measured as an index of adenyl cyclase activity. The data showed that both the ability to incorporate [3H] into ATP and adenyl cyclase activity were significantly lower in psoriatic plaques than in uninvolved skin of the psoriatic patients, or in normal skin of control subjects. The response of adenyl cyclase to the stimulation of 3.3 muM adrenaline was less than five fold in psoriatic plaques as compared to twelve to thirty-two fold in the uninvolved skin. The response to the stimulation of prostaglandin E2 (5 mug/ml) showed no significant difference between the plaque and normal skin. The adenyl cyclase activity in uninvolved skin of psoriatic patients appeared normal. Propranolol (10 muM) blocked the stimulatory effect of adrenaline but not that of PGE2 in normal skin. These results suggest that the adenyl cyclase system of the skin has different regulatory sites for adrenaline and PGE2 and that the enzyme is defective in the epidermis of the psoriatic plaque, especially at the adrenaline regulatory site.