Visualization of gap junction mobility in living cells.

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.

Occurrence of positive immunofluorescence in the dermo-epidermal junction of sun-exposed skin of normal adults.

A bright, continuous, granular deposition of immunoreactants at the dermo-epidermal junction (DEJ) of lesional skin is highly suggestive of cutaneous lupus erythematosus (LE). A recent study of the direct immunofluorescence (IF) of sun-exposed skin in normal adults has demonstrated findings similar to the bright, continuous granular pattern found in cutaneous LE. This data suggests that positive IF from sun-exposed cutaneous lupus lesions is nonspecific. Forty-one healthy adults, without a history of dermatoses or photosensitivity, presenting to the dermatology clinic for the excision of skin cancers were studied. Excess non-lesional tissue, removed from Moh's excision sites (sun-exposed face and neck) in order to obtain appropriate cosmetic closure, was examined for the deposition of immunoreactants. The specimens were incubated with fluoresceinated monovalent anti-human immunoglobulin specific for IgG, IgA, IgM, C3, Clq, and fibrinogen and examined independently by 2 immunodermatologists without prior knowledge of patient or site. None of the samples demonstrated immunoreactant deposition consistent with cutaneous LE. IF of several specimens (21/41) had a weak (1+ or 2+), interrupted pattern of fibrinogen at the DEJ,--a common, non-specific finding. Weak, interrupted, linear and granular patterns were seen with IgM (10/41), Clq (9/41), IgG (2/41), IgA (2/41), and C3 (1/41). Fibrinogen was the only immunoreactant demonstrating a bright (3+), continuous, granular pattern (4/41). This data suggests that sun-exposure alone does not induce the deposition of immunoreactants at the DEJ in a pattern similar to that found in cutaneous LE.

Co-localization of proenkephalin- and prodynorphin-derived opioid peptides in laminae IV/V spinal neurons revealed in arthritic rats.

By the use of highly selective antisera and an immunohistochemical technique the possible coexistence of proenkephalin- (PRO-ENK)- and prodynorphin (PRO-DYN)-derived peptides was examined in 4- to 6-micron thick serial sections of the L4-L5 segments of the spinal cord of non-colchicine-treated polyarthritic rats. In control, non-colchicine treated animals, virtually no cell bodies stained for the PRO-ENK-derived peptides, heptapeptide (MRF) and octapeptide (MRGL), nor for the PRO-DYN-derived peptides, dynorphin A (DYN) and alpha-neoendorphin (NEO). In contrast, in polyarthritic rats, numerous large (15-30 micron) multipolar neurons could be visualized with each antiserum in laminae IV/V. Alternate staining of adjacent sections with either anti-MRF or anti-MRGL antisera, followed by either anti-DYN or anti-NEO antisera, revealed a clear coexistence of PRO-ENK and PRO-DYN peptides. It was possible to demonstrate co-localization of all 4 opioids in a single cell. It appeared that all cells staining for PRO-ENK peptides in laminae IV/V also stained for PRO-DYN peptides.

Co-existence of prodynorphin--opioid peptides and substance P in primary sensory afferents of guinea-pigs.

Light microscopic immunoenzymatic and immunofluorescence histochemistry revealed co-existence of opioid peptides with substance P in primary sensory neurons of all segmental dorsal root and trigeminal ganglia of guinea-pig. Sensory opioid peptides appeared to be exclusively processed from prodynorphin and include [Leu]enkephalin, neoendorphins, and dynorphin A. We suggest the presence of presynaptic opioid autoreceptors in primary sensory afferences.