The human placenta does not contain lipofuscin pigment.

Autofluorescent granules are present in the villous syncytiotrophoblast of the human placenta, most prominently during the first trimester. These granules differ in both size and hue from classical lipofuscin granules, have different staining characteristics, and are shown by electron microscopy to be syncytial lipid droplets. It is concluded that previous reports of the presence of lipofuscin pigment in the placenta are unfounded.

Zinc-containing fiber systems in the cochlear nuclei of the rat and mouse.

Zinc-containing neurons are cells which sequester zinc in the vesicles of their axonal boutons; such zinc-containing fiber systems have been previously shown to innervate many limbic and cerebrocortical brain regions. The present study of rats and mice shows that zinc-containing axons also innervate the cochlear nuclei, forming two morphologically-distinct projection systems. One zinc-containing pathway innervates the molecular stratum of the dorsal nucleus, supplying a diffuse, even band of neuropil staining throughout the stratum. The other pathway projects sparsely to the various small cell (granule cell) regions of the nuclei where the zinc-positive elements form scattered clusters and threads of bouton-like puncta amidst the granule neuron somata. Preliminary observations indicate that the pattern is the same in the cat as in the rat and mouse.

'Small'-proteoglycan:collagen interactions: keratan sulphate proteoglycan associates with rabbit corneal collagen fibrils at the 'a' and 'c' bands.

Proteoglycans (PGs) in rabbit corneal stroma and mouse sclera have been stained for electron microscopy with Cupromeronic blue in a critical electrolyte concentration (CEC) mode, with and without prior digestion of the tissue by keratanase or chondroitinase ABC to remove the keratan sulphate (KS) or chondroitin-dermatan sulphates (CS or DS) respectively. Two classes of PGs, located orthogonally to the corneal collagen fibrils at either the 'step' (band 'a' or 'c') or gap zone (band 'd' or 'e') are shown to be KS-PGs or DS-PGs respectively. Four separate and specific PG binding sites on Type I collagen fibrils have thus been identified. Rabbit corneal KS and DS PGs each contain two kinds of PG (Gregory JD, Coster L & Damle SP (1982) J. Biol. Chem. 257, 6965-6970). We propose that each 'small' protein-rich PG is associated with a specific binding site on the collagen fibril.

Proteoglycan-type I collagen fibril interactions in bone and non-calcifying connective tissues.

The association of proteoglycans with type I collagen fibrils in skin, tendon, cornea and bone has been determined by electron microscopy using an electron-dense dye, Cupromeronic blue, in the critical electrolyte concentration mode, backed up by biochemical analysis and digestion by hyaluronidase or keratanase. A major proteoglycan of the soft tissues, containing dermatan sulphate, is shown to be regularly and orthogonally arranged at the surface of the fibrils. Uranyl acetate counterstaining revealed that the main specific binding site is the 'd' band, which previous work indicated is very close to the initial site of calcification of type I collagen fibrils. Bone, demineralized by a 'non-aqueous' technique which preserves the proteoglycan in the tissue, does not contain orthogonal arrays; the interfibrillar proteoglycan filaments are oriented parallel to the fibril axis. The main proteoglycan in bone is chondroitin sulphate-rich. It is suggested that dermatan sulphate proteoglycan plays a role in preventing soft connective tissues from calcifying.

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands.

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a-e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep. 5:71-81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.

Identification of specific binding sites for keratan sulphate proteoglycans and chondroitin-dermatan sulphate proteoglycans on collagen fibrils in cornea by the use of cupromeronic blue in 'critical-electrolyte-concentration' techniques.

Proteoglycans (PGs) in bovine corneal stroma were stained with Cupromeronic Blue in 'critical-electrolyte-concentration' (CEC) methods for electron microscopy, and were located vis-à-vis collagen fibril a-e banding patterns. Keratanase and chondroitin ABC lyase digestion showed that a + c-band- and d + e-band-associated PGs were keratan sulphate-rich and chondroitin (dermatan) sulphate-rich respectively. The CEC pattern proved that the keratan sulphate PGs at the a and c bands differed. Comparison of their CECs with their behaviour on anion-exchange chromatography confirmed previous (indirect) attempts at identification [Scott & Haigh (1985) Biosci. Rep. 5, 765-774]. Similar arguments were applied to the dermatan sulphate PGs at the d and e bands. These results strongly support the one-PG-one-binding-site hypothesis [e.g. Scott (1988) Biochem. J. 252, 313-323]. Remarkable inter-species variations in the keratan sulphate PG patterns contrast with the relatively constant picture of dermatan sulphate PG-collagen fibril interactions.

Keratan sulphate and the ultrastructure of cornea and cartilage: a 'stand-in' for chondroitin sulphate in conditions of oxygen lack?

Corneas from mouse, rat and rabbit were analysed quantitatively and/or qualitatively for collagen and acid glycosaminoglycans. They were examined by light and electron microscopy, using Alcian blue and Cupromeronic blue, in critical electrolyte concentration methods, with or without digestion by hyaluronidase, chondroitinases and keratanase, for their sulphated glycosaminoglycan distributions. Glycosaminoglycan patterns were very different in the three species. Mouse lacked chemically detectable keratan sulphate, which was present in considerable amounts in rat and rabbit stroma. Mouse corneal stroma proteoglycan filaments were located predominantly at the gap zone of the collagen fibrils, mainly at the d band, with few at the a and c bands. Rat and rabbit micrographs were more complicated, with many proteoglycan filaments at the a and c, as well as the d and e bands. These findings support the proposal that the a and c bands were specific binding sites for keratan sulphate proteoglycan (Scott & Haigh, 1985b). Evidence from studies on cornea and cartilage suggests that keratan sulphate, rather than chondroitin sulphate is produced in conditions of O2 lack. Metabolic mechanisms which could account for this balance are proposed The production of uridine diphosphate glucuronic acid is the key step, which is sensitive to hypoxia, lactate and NAD:NADH ratios.

A method of processing tissue sections for staining with cu-promeronic blue and other dyes, using CEC techniques, for light and electron microscopy.

The application to connective tissues etc. of new histochemical and ultrastructural methods involving consecutive treatment with dye-salt baths, specific enzymes, antibodies and decalcifying fluids, is best carried out on sections on glass slides. This ensures uniform access of reagents to substrates and allows the accurate monitoring of the results, as well as exploiting the strengths of the newer reagents (e.g. Cupromeronic Blue) in that they are highly coloured as well as electron dense. However, very high losses of connective tissue sections usually occur in the various solutions, to the extent that many important investigations are rendered almost impossible because of the high cost of the reagents. We describe a technique which we use routinely, that gives very high recovery of sections of "difficult" tissues from multi-stage enzyme and stain treatments, for light microscopy followed by plastic embedding of the coloured sections for electron microscopy.

The effect of muscle paralysis on the radial growth of collagen fibrils in developing tendon.

Voluntary muscle activity in chick embryos was paralysed by administration in ovo of tubocurarine hydrochloride, administered in single or multiple doses, from day 9 to day 13 after fertilization. Control eggs were given saline instead of tubocurarine or were simply incubated without operative interference. Embryos were killed at 9, 13, 14, 16 and 19 days after fertilization. Flexor digitorum tendons were removed, fixed in glutaraldehyde, embedded in plastic, sectioned, and stained with phosphotungstic acid for electron microscopy. The diameters of the tendon collagen fibrils were measured, on electron micrographs, using a Magiscan Mk II programme. Tendon collagen fibril expansion was not inhibited by tubocurarine treatment. It is concluded that the rapid increase of collagen fibril diameters, which coincides in the normal embryo with the first onset of use of the associated muscle, is not dependent on muscle activity. There remains a possibility that other ways of producing tension in the tendon could provide sufficient stimulus to fibril expansion.

Proteoglycan: collagen interactions in dermatosparactic skin and tendon. An electron histochemical study using cupromeronic blue in a critical electrolyte concentration method.

Proteoglycans (PGs), stained for electron microscopy with Cupromeronic blue, were observed in skin and tendon from normal and dermatosparactic calves. Very frequently they (i.e. dermatan sulphate (DS) PGs) were seen arrayed orthogonally to the collagen fibrils, in the gap zone, usually at the d band, in both diseased and normal tissues. Where UO2(2+) staining showed regular and normal packing of collagen molecules, orthogonally located DS PGs were seen. No qualitative differences between controls and pathological tissues were identified, but quantitatively it appears likely that considerable areas of the surface of dermatosparactic skin collagen fibrils may be without associated PGs.

Laser flash photolysis of diphenylsulfonyldiazomethane: detection of the sulfene and a sulfene-pyridine ylide

The photochemistry of diphenylsulfonyldiazomethane (DSD) was studied by means of nanosecond laser flash photolysis. The photochemical behavior of this molecule upon UV irradiation is characterized by sulfene formation, presumed to arise via Wolff rearrangement of a carbene. We were able to detect the sulfene and the sulfene ylide formed upon sulfene trapping by pyridine. Sulfene quenching by nucleophiles was also examined.