Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca2+ medium (0.08 mM) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56 +/- 0.05), steadily increased to a maximum of 0.84 +/- 0.09 between Days 2 and 4, and then gradually decreased to 0.41 +/- 0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [3H]thymidine into their nuclei. Electrophoresis of [3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of "96K-associated UEA-bound" radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis.     

Defining the identity of mouse embryonic dermal fibroblasts

Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase‐seq and histone modification ChiP‐seq data on various cell‐types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell‐types. We found a subset of the signature genes whose expression is dependent on Wnt/β‐catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415–430, 2016. © 2016 Wiley Periodicals, Inc.     

Application of an Optimized Tape Stripping Method for the Bioequivalence Assessment of Topical Acyclovir Creams

This study indicates the application of tape stripping (TS) for bioequivalence (BE) assessment of a topical cream product containing 5% acyclovir. A TS method, previously used successfully to assess BE of topical clobetasol propionate and clotrimazole formulations, was used to assess BE of an acyclovir cream (5%) formulation as well as a diluted acyclovir formulation (1.5%) applied to the skin of healthy humans. An appropriate application time was established by conducting a dose duration study using the innovator product, Zovirax® cream. Transepidermal water loss was measured and used to normalize thicknesses between subjects. The area under the curve (AUC) from a plot of amount of acyclovir/strip vs cumulative fraction of stratum corneum (SC) removed was calculated for each application site. BE was assessed using Fieller’s theorem in accordance with FDA’s guidance for assessment of BE of topical corticosteroids. Adco-acyclovir cream (5%) was found to be BE to Zovirax® cream, where the mean test/reference (T/R) ratio of the AUC’s was 0.96 and the bioequivalence interval using a 90% confidence interval was 0.91–1.01 with a statistical power >?95%, whereas the diluted test product fell outside the BE acceptance criteria with T/R ratio of AUC of 0.23 and a 90% CI of 0.20–0.26. This study indicates that the data resulting from the application of this TS procedure has reinforced the potential for its use to assess BE of topical drug products intended for local action, thereby obviating the necessity to undertake clinical trials in patients.     

The Diagnostic Value of Serum Leucine Aminopeptidase

Experience with serum leucine aminopeptidase determination in 61 patients led to the following conclusions. The test is no more sensitive than the alkaline phosphatase test in cases of carcinoma of the pancreas. It is elevated in all cases of obstructive and hepatogenous jaundice and serves no useful function in their differentiation. It was a valuable test in cases of calculous biliary tract disease, being more sensitive than either bilirubin or alkaline phosphatase determinations. In three instances of elevated leucine aminopeptidase in disorders apparently not related to the liver or pancreas, laparotomy and autopsy showed involvement of these organs in two.     

Biosynthesis of Phenylalanine from Phenylacetate by Chromatium and Rhodospirillum rubrum

Cultures of Chromatium strain D and Rhodospirillum rubrum incorporated ¹⁴C from phenylacetate-1-¹⁴C during anaerobic growth. The radioactivity in the protein fraction of cells was mainly in phenylalanine. Phenylalanine from Chromatium cells grown in phenylacetate-1-¹⁴C was labeled at carbon 2. Incorporation of phenylacetate by Chromatium was decreased in the presence of exogenous phenylalanine, and de novo synthesis of phenylalanine from bicarbonate was less in medium containing either phenylalanine or phenylacetate. These organisms, and also certain anaerobic rumen bacteria, apparently carboxylate phenylacetate to synthesize the phenylalanine carbon skeleton. The mechanism of the carboxylation is unknown; however, it appears to be dependent upon anaerobic conditions, since R. rubrum did not synthesize phenylalanine from phenylacetate during aerobic growth in the dark.     

The Use of Salicylic Acid as a Fixative

The following salicylic acid-containing fixatives are useful for cytological studies in plants. The first, here designated HFC, is recommended for studies on somatic mitosis and chromosome individuality. The second, denoted HFP, is recommended for studies on plastids.

HFC is made up in two solutions. Sol. A: 100 cc. sat. aq. sol. salicylic acid, slight excess copper hydroxide, 20 cc. formaldehyde, 30 cc. normal ortho-phosphoric acid, 200 cc. water, 1 g. saponin; pH 1.8 to 1.9. Fix in Sol. A 15 to 30 minutes in partial vacuum of 35 cms. Then add Sol. B: 1% aq. chromic acid in equal parts. Continue fixation for period of 18 to 24 hours.

HFP is also made up in two solutions which are used in equal parts. Sol. A: 100 cc. sat. aq. sol. salicylic acid, slight excess copper hydroxide, 10 cc. normal ortho-phosphoric acid, 1/2 g. saponin. Sol. B: 187.5 cc. 1% aq. chromic acid, 50 cc. 2% osmic acid. Fixation technic as HFC.

Dehydrate and infiltrate with paraffin after Zirkle. Stain with crystal-violet-iodine.     

Retinoids alter the direction of differentiation in primary cultures of cutaneous keratinocytes

The effects of vitamin A on the morphological expression of differentiation were studied in cell cultures of cutaneous keratinocytes from the newborn rat. The cells were first cultivated in a medium containing 0.11 mM calcium until a confluent monolayer had been formed. Stratification and terminal differentiation were then triggered by raising the calcium concentration of the medium to 1.96 mM ('normal' culture). The rise in the concentration of calcium was coupled with the addition of retinol (RL) of retinoic acid (RAC) to the medium to produce an excess of vitamin A (high-retinoid culture). Delipidized serum was used to produce a deficiency of vitamin A (low-retinoid culture). The tissue organization and the ultrastructure of the keratinocytes in the stratified culture were the same as those seen in conventional cultures and skin explants. These stratified cultures expressed the morphological features of the epidermis of intact skin. The addition of RL or RAC to the medium enhanced features characteristic of the secretory epithelium, such as the formation of an extensive endoplasmic reticulum, an enlargement of the Golgi zone, and an increase in the number of vacuoles. At the same time, the addition of retinoids diminished features characteristic of the terminal differentiation of the stratified squamous epithelium, such as stratification and keratinization. Deficiency of vitamin A in the medium resulted in a culture with many differentiated layers. The differentiated cells of the low-retinoid cultures contained densely packed tonofilaments and synthesized products that reacted with the monoclonal antibody AE2 that is specific for keratin peptides which are markers of epidermal differentiation. In the cell culture system that is presented here, an excess of retinoids redirected epithelial differentiation from a stratifying and keratinizing epithelium towards a secretory epithelium. This system is a useful tool for elucidating the mechanisms responsible for the effect of vitamin A on the differentiation of epithelial cells.     

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10^-5 M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of the cells with a microfilament-disrupting drug, cytochalasin B (10 g/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5–15 min. A morphological abnormality, microtubule paracystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.     

In Vitro Dissolution of Generic Immediate-Release Solid Oral Dosage Forms Containing BCS Class I Drugs: Comparative Assessment of Metronidazole,Zidovudine, and Amoxicillin Versus Relevant Comparator Pharmaceutical Products in South Africa and India

Biowaivers are recommended for immediate-release solid oral dosage forms using dissolution testing as a surrogate for in vivo bioequivalence studies. Several guidance are currently available (the World Health Organization (WHO), the US FDA, and the EMEA) where the conditions are described. In this study, definitions, criteria, and methodologies according to the WHO have been applied. The dissolution performances of immediate-release metronidazole, zidovudine, and amoxicillin products purchased in South African and Indian markets were compared to the relevant comparator pharmaceutical product (CPP)/reference product. The dissolution performances were studied using US Pharmacopeia (USP) apparatus 2 (paddle) set at 75 rpm in each of three dissolution media (pH1.2, 4.5, and 6.8). Concentrations of metronidazole, zidovudine, and amoxicillin in each dissolution media were determined by HPLC. Of the 11 metronidazole products tested, only 8 could be considered as very rapidly dissolving products as defined by the WHO, whereas 2 of those products could be considered as rapidly dissolving products but did not comply with the f₂ acceptance criteria in pH 6.8. All 11 zidovudine products were very rapidly dissolving, whereas in the case of the 14 amoxicillin products tested, none of those products met any of the WHO criteria. This study indicates that not all generic products containing the same biopharmaceutics classification system (BCS) I drug and in similar strength and dosage form are necessarily in vitro equivalent. Hence, there is a need for ongoing market surveillance to determine whether marketed generic products containing BCS I drugs meet the release requirements to confirm their in vitro bioequivalence to the respective reference product.KEY WORDS: BCS, dissolution testing, generic drug, immediate-release solid oral dosage forms, WHO criteria