Isolation of two immunologically related transglutaminase substrates from cultured human keratinocytes

Transglutaminase substrates A and B were identified in soluble extracts of cultured keratinocytes and human epidermis by their reactions with dansyl cadaverine in the presence of Ca++ ion. Substrate B was present in substantial amounts in both extracts whereas A, easily seen in cell extracts, was decreased and sometimes not detected in tissue extracts. Substrates A and B from cultured cells were separated by Sephadex G-75 chromatography and isolated by preparative sodium dodecyl sulfate (SDS) gel electrophoresis by which A had a mol wt of 125,000 and B had a mol wt of 12,000. Amino acid analysis of A, B, and cornified envelope were similar but not identical. The isopeptide bond is not a significant structural feature of A inasmuch as its content is less than 0.25 bonds/molecule. Antibodies raised to A cross-reacted with B and vice versa and A showed partial identity to B when reacted with anti-B. Anti-A reacted with epidermis being adsorbed by the edges of cornifying cells but only weakly by cells of the Malpighian layer. Anti-B also reacted with cornifying, but its reaction was more intense with the cytoplasm of Malpighian cells. Substrate A appears to be incorporated into cornified envelope immediately after its appearance in cells of the granular layer and seems similar to a protein isolated by a different method. Substrate B, convertible by transglutaminase to higher molecular weight species, may also participate in cornified envelope assembly and shares some structural similarities to A.     

Identification and characterization of a previously undetected region between the perichondrium and periosteum of the developing avian limb

In developing long bones, the growing cartilage and bone are surrounded by the fibrous perichondrium (PC) and periosteum (PO), respectively, which provide cells for the appositional growth (i.e., growth in diameter) of these tissues. Also during the longitudinal growth of a bone, the cartilage is continuously replaced by bony tissue, giving rise to the widely held assumption that the PC concomitantly gives rise to the PO. Except for this morphological correlate, however, no evidence exists for a direct conversion of PC cells to PO cells, and our observations presented here question this assumption. Instead, we have obtained evidence suggesting that a previously undescribed region exists between the PC and PO. This region, termed the border region (BR), has several unique characteristics which distinguish it from either the PC or PO, including (1) its lack of being determined to differentiate as either cartilage or bone, (2) its ability to preferentially elicit the invasion of blood vessels, and (3) its ability to undergo preferential growth.     

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.     

The pancornulins: a group of basic low molecular weight proteins in mammalian epidermis and epithelium that may function as cornified envelope precursors

1. A monoclonal antibody (HCE-2) to human epidermal and epithelial cornified envelopes identified a group of soluble basic protein precursors. 2. Using HCE-2, envelope-like staining was observed in the epidermis and stratified squamous epithelium of a number of mammalian species. 3. Basic polypeptides reactive to HCE-2 varied in size and number among the different animals. 4. In those species studied, HCE-2-reactive peptides were substrates for transglutaminase and protease treatment of cornified envelopes released HCE-2-reactive degradation products. 5. These results suggest a new family of proteins in mammalian epidermis that may function as cornified envelope precursors.     

Cortisol stress response and histopathological alteration index in Clarias gariepinus exposed to sublethal concentrations of Qua Iboe crude oil and rig wash

The histological effect on and stress response of post juvenile Clarias gariepinus exposed to Qua Iboe crude oil and rig wash were investigated. Fish weighing 60–90 g and measuring 16–18 cm were exposed for 7–28 days to 8.00 ml^?1 Qua Iboe crude oil and 0.0018 ml^–1 rig wash, both being 0.1 of the 96 hr LC₅₀. Blood samples of C. gariepinus were collected every seven days and evaluated for stress by measuring cortisol concentration. The gills and liver were studied and scored for Gill Alteration Index (GAI) and Hepatic Alteration Index (HAI), respectively. There was an increase in cortisol level up to the 7th and 14th day among the group exposed to Qua Iboe crude oil, with a decrease on the 21st and 28th day. The rig wash group increased in cortisol level up to the 7th day and decreased slightly on the 14th day, after which the trend became irregular. The toxic effects of the Qua Iboe crude oil and rig wash were time dependent, as shown by the histopathological alteration index (HAI) of gill and liver. After 28 days of exposure, the gills had irreparable damage due to high frequency of cellular necrosis and degeneration, whereas the liver had from moderate to severe damage due to the high frequency of cellular degeneration and inflammation. Qua Iboe crude oil and rig wash are both toxic to C. gariepinus, therefore their indiscriminate discharge to the environment must be discouraged.     

Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r2) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.