Coordinate assembly of lipids and enzyme proteins into epidermal lamellar bodies.

Formation of the epidermal permeability barrier requires delivery of lamellar body (LB) contents to the stratum corneum interstices. LB are enriched in a mixture of polar lipids and a family of hydrolytic enzymes, required for the extracellular processing of the secreted polar lipids into the more hydrophobic products which mediate barrier function. Prior non-quantitative studies show that acute barrier disruption leads to immediate secretion of the contents of performed LB from the outermost layer of granular cells, followed by the synthesis and accelerated secretion of newly-formed (= nascent) organelles over 0.5-4 h. We asked here whether lipids and hydrolytic enzymes are packaged into nascent organelles separately, or in a parallel, linked process. We first quantified the rate of appearance of lipids (by the content of internal lamellae within LB) and enzyme content (by cytochemistry of neutral lipase and acid sphingomyelinase); both are concentrated in LB, and in nascent organelles. Immediately after barrier disruption, the density of LB in the cytosol of the outermost granular cell decreased by > 50% reduction at 30 min, returning to near-normal densities by 4 h. Nascent organelles budded off a trans-Golgi-like reticulum, in the outermost granular cells as early as 30 min. In quantitative studies, LB progressively accumulated lipid and enzyme contents in parallel. However, when lipid/lamellae generation was inhibited with lipid synthesis inhibitors, enzymes did not accumulate in organelles. Likewise, when exogenous physiologic lipids were delivered to sites of LB generation in the face of brefeldin A blockade of organellogenesis, or when lipids were delivered in conjunction with treatment with lipid synthesis inhibitors, enzymes accumulated only in those organelles that displayed lipid content. These studies demonstrate: (a) quantitative changes in the density of LB in the outermost granular cell at various time points after acute barrier disruption; (b) the origin of nascent organelles in a trans-Golgi-like reticulum; (c) co-ordinate packaging of lipid and enzyme contents into nascent organelles; (d) that lipid deposition in nascent organelles is required for enzyme accumulation; and (e) that enzymes can be delivered to nascent organelles, even if the source of lipid is of exogenous rather than endogenous origin.

Microwave incubation improves lipolytic enzyme preservation for ultrastructural cytochemistry.

Standard methods for the ultrastructural detection of lipase and sphingomyelinase activities in the skin result in considerable loss of structural preservation, often interfering with accurate delineation of enzyme localization in association with specific organelles. Moreover, poor preservation occurs, even after extensive aldehyde prefixation, owing to the prolonged incubation times needed to detect residual enzyme activity, which often require non-physiological conditions. A modified incubation protocol is described here, which uses microwave irradiation in conjunction with drastically shortened incubation times, resulting in both superior ultrastructural preservation and excellent localization in mammalian epidermis. This method should be useful generally not only for the study of lipase localization in skin, but also in conjunction with the cytochemical detection of a variety of enzymes in various types of tissue.

Evidence that the gonadotrophs are the likely site of production of angiotensin II in the anterior pituitary of the rat.

To determine whether the angiotensin II-like immunoreactivity (AII-IR) previously reported in rat gonadotrophs is generated locally, we stained for AII-IR in neonatal rat pituitary explants and dispersed adult pituitary cells maintained in serum-free medium. In both preparations, AII-IR was found in cells with the morphological characteristics of gonadotrophs. In the anterior pituitaries of adult male rats, AII-IR and LH beta immunoreactivity were found by electron microscopy to be located in the same secretory granules. Pituitary tissue was also extracted, and the AII content was measured by RIA. The gland contained 300 times more AII than could be accounted for by the extracellular fluid in the gland. On HPLC, the pituitary AII-IR comigrated with synthetic Ile5-AII. Thus, it appears that the AII-IR in rat pituitary gonadotrophs is AII and that it is likely to be produced within these cells.

Lowicryl K4M embedding of brain tissue for immunogold electron microscopy.

We present methods for embedding brain tissue in Lowicryl K4M embedding medium and localizing antigens using postembedding immunogold techniques. After perfusion fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, blocks of rat brain were placed in 2% aqueous uranyl acetate for 1 hour, dehydrated in 50%, 70%, and 95% ethanol, infiltrated with Lowicryl/ethanol mixtures (1:2 for 10 min, 1:1 for 15 min) and 100% Lowicryl (20 min and 25 min). Polymerization was carried out under UV light for 24-48 hours at room temperature. Several neural antigens, including three different synaptic vesicle proteins and an enzyme associated with the postsynaptic density, were localized by this technique, indicating that this procedure may have wide applicability.

Development of arthrospores of Trichophyton mentagrophytes.

Arthrosporogenesis of the dermatophyte Trichophyton mentagrophytes was examined by light and by scanning and transmission electron microscopy. Sabouraud dextrose agar plates were inoculated with microconidia and incubated in an atmosphere of 8% CO2. Typical germination and hyphal branching continued to day 4, when hyphae began to be increasingly coated with granular-fibrillar material. Multiple replication of nuclei and formation of segregating septa followed. By day 6 the thick surface mesh sometimes was restricted to protruding rings, probably over septa. Between days 6 and 7, after thickening of outer and septal walls, units began to round and separate. Triangular gaps, which developed at the junction of septa and outer wall layers, enlarged so that spores were held together at their poles and along a tangential ring. With elongation of the spore to its barrel shape, the halves of the septum separated and the ring pulled apart, leaving a jagged, circular flange originating from the outer layer of cell wall extended toward the poles, covering the apparently exposed inner wall layer. Newly formed arthrospores, which measured 2.0 to 3.3 by 2.9 to 3.8 micronm and possessed walls of about 0.33-micronm thickness, has smooth sides but somewhat rough poles.