The lead hydroxide stain of Watson (1958) used for increasing contrast in thin sections for electron microscopy has found acceptance in many laboratories. However, this stain has an unfortunate tendency to form precipitates (probably of lead carbonate (5)) on exposure to the air, thus contaminating the sections and irritating the observer. This drawback has led to the development of several modifications (2, 3) of the original method of staining and the use of ingenious devices (4, 5) for preventing exposure to air and consequent precipitate formation. We offer the following alternative methods which, we believe, are simpler to perform than those hitherto described. They have the additional advantages mentioned below. The methods are based on the observation that highly alkaline solutions of lead salts (pH > 11.5) yield relatively stable solutions which stain rapidly and intensely, thus obviating the hazard of precipitation to a marked degree. The methods have these additional advantages: the staining solutions are easily and rapidly prepared, are simply stored, and are stable for long periods of time. Furthermore, they can be efficiently used, many grids being treated simultaneously, without excessive precautions being taken against lead carbonate precipitation. Finally, "difficult" material, embedded in media which characteristically yield rather low contrast, such as epoxide resins, can be rapidly and easily stained. "C lean" preparations, of high contrast, are routinely obtained. As will be discussed later, it is thought that in these highly alkaline staining solutions lead is present as an hydroxide complex anion (plumbite ion) and that this anion is responsible for the staining. The methods of preparation are based on this hypothesis. Two methods for preparing the staining solutions have been found useful:
Acidosis of the tumor microenvironment is typical of a malignant phenotype, particularly in hypoxic tumors. All cells express multiple isoforms of carbonic anhydrase (CA), enzymes catalyzing the reversible hydration of carbon dioxide into bicarbonate and protons. Tumor cells express membrane-bound CAIX and CAXII that are controlled via the hypoxia-inducible factor (HIF). Despite the recognition that tumor expression of HIF-1alpha and CAIX correlates with poor patient survival, the role of CAIX and CAXII in tumor growth is not fully resolved. To understand the advantage that tumor cells derive from expression of both CAIX and CAXII, we set up experiments to either force or invalidate the expression of these enzymes. In hypoxic LS174Tr tumor cells expressing either one or both CA isoforms, we show that (a) in response to a "CO(2) load," both CAs contribute to extracellular acidification and (b) both contribute to maintain a more alkaline resting intracellular pH (pH(i)), an action that preserves ATP levels and cell survival in a range of acidic outside pH (6.0-6.8) and low bicarbonate medium. In vivo experiments show that ca9 silencing alone leads to a 40% reduction in xenograft tumor volume with up-regulation of ca12 mRNA levels, whereas invalidation of both CAIX and CAXII gives an impressive 85% reduction. Thus, hypoxia-induced CAIX and CAXII are major tumor prosurvival pH(i)-regulating enzymes, and their combined targeting shows that they hold potential as anticancer targets.
The ‘acid mantle’ of the stratum corneum seems to be important for both permeability barrier formation and cutaneous antimicrobial defense. However, the origin of the acidic pH, measurable on the skin surface, remains conjectural. Passive and active influencing factors have been proposed, e.g. eccrine and sebaceous secretions as well as proton pumps. In recent years, numerous investigations have been published focusing on the changes in the pH of the deeper layers of the stratum corneum, as well as on the influence of physiological and pathological factors. The pH of the skin follows a sharp gradient across the stratum corneum, which is suspected to be important in controlling enzymatic activities and skin renewal. The skin pH is affected by a great number of endogenous factors, e.g. skin moisture, sweat, sebum, anatomic site, genetic predisposition and age. In addition, exogenous factors like detergents, application of cosmetic products, occlusive dressings as well as topical antibiotics may influence the skin pH. Changes in the pH are reported to play a role in the pathogenesis of skin diseases like irritant contact dermatitis, atopic dermatitis, ichthyosis, acne vulgaris and Candida albicans infections. Therefore, the use of skin cleansing agents, especially synthetic detergents with a pH of about 5.5, may be of relevance in the prevention and treatment of those skin diseases.
AbstractPolypseudorotaxane (PPR) hydrogels formed by inclusion complexes between poly(ethylene glycol) (PEG) and α‐cyclodextrin (α‐CD) are highlighted as promising biomaterial for drug delivery. Here, we report a novel injectable PPR hydrogel containing graphene oxide (GO) for pH‐responsive controlled release of doxorubicin hydrochloride (DOX). Our results showed that the gelation rates of the PEG/α‐CD supramolecular structures could be tailored depending on the reagent concentrations. The formation of PEG/α‐CD inclusion complexes was confirmed by TEM and XRD, the latter further confirming that GO restricts their formation. The supramolecular hydrogels were easily loaded with DOX by simple addition into the PEG solution before the complex formation with the α‐CD solution. Noteworthy, disruption of ionic interactions between DOX and GO in the nanocomposite at pH = 5.5 resulted in higher DOX release than under physiological conditions (pH = 7.4). This pH dependence was barely observed in pure PPR hydrogel. These findings introduce DOX‐loaded supramolecular hydrogels nanocomposites as promising carriers for pH‐responsive and therefore localized, drug delivery systems.