Immunoelectron microscopy of rhodopsin and vitamin A in the pineal organ and lateral eye of the lamprey.

Rhodopsin- and vitamin A-immunoreactive sites were studied in the pineal organ of the larval and adult brook lamprey (Lampetra planeri Bloch), as well as in the retina of the larval lateral eye, at the electron microscopic level. In the pineal organ, several types of photoreceptor cells could be distinguished by their morphology and the immunoreactivity of their outer segments. The different kinds of photoreceptor cells were located at different levels of the pineal organ according to their distance from the "pineal window". The most superficial level, the so-called pellucida, appears to represent an exclusively "cone-type" area containing slender, rhodopsin-immunonegative (UV-blue-sensitive?) photoreceptors only. The second level, the pineal retina, contained predominantly rod-type photoreceptors, i.e., large, strongly rhodopsin-immunopositive (green-sensitive) photoreceptors medially, and few, small, weakly rhodopsin-immunopositive (blue-green-sensitive?) cells bilaterally. At the deepest level, the pineal atrium, there were both rod- and cone-type photoreceptor cells, the latter possibly representing red-sensitive elements. Vitamin A immunoreactivity was found in the outer segments of the pineal photoreceptor cells, in the cytoplasm and mitochondria of inner segments and perikarya, as well as in nuclear euchromatin and compact nucleoli. A similar gold labelling of organelles was observed in the ependyma and pineal neurons. The vitamin A immunoreaction of the outer segments suggests retinoids are present as chromophores of the photopigments. In the peripheral retina of the larval lateral eye, vitamin A immunoreactivity was found in some organelles of the undifferentiated photoreceptor cells, neurons, pigment epithelium and Müllerian cells. The localization of immunoreactive vitamin A in nuclei, nucleoli and cytoplasm including mitochondria appears to strengthen the case for an interaction of retinoids in the function of these organelles.

Immunocytochemical localization of vitamin A in the retina and pineal organ of the frog, Rana esculenta.

Vitamin A immunoreactive sites were studied in the retina and pineal organ of the frog, Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. In dark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. In light-adapted retina, cones and green (blue-sensitive) rods were immunopositive. At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photorecptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments. The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.

The first component of complement as a constituent of human salivary sediment.

The sediment from human saliva is complement-reactive. Evidence presented shows that C1 (first component of complement) is a constituent of sediment from healthy human donors. Sediment (Sed) inactivated functionally pure C4 (fourth component of complement), and this action on C4 was inhibited by EDTA, phenylmethylsulphonylfluoride (PMSF, a serine-esterase inhibitor) and C1-inhibitor (C1-In). When Sed was incubated with 0.15 ionic strength buffer and separated by centrifugation, C1 haemolytic activity was found in the supernatant. By incubating Sed with EAC4 cells (sheep erythrocytes sensitized with rabbit antibody to which C4 has been fixed), transfer was shown of C1 from the Sed to the cells, resulting in the formation of EAC14; this transfer was inhibited by IgG directed against a subunit of C1 (anti-C1s).

Carbohydrate and protein contents of grain dusts in relation to dust morphology.

Grain dusts contain a variety of materials which are potentially hazardous to the health of workers in the grain industry. Because the characterization of grain dusts is incomplete, we are defining the botanical, chemical, and microbial contents of several grain dusts collected from grain elevators in the Duluth-Superior regions of the U.S. Here, we report certain of the carbohydrate and protein contents of dusts in relation to dust morphology. Examination of the gross morphologies of the dusts revealed that, except for corn, each dust contained either husk or pericarp (seed coat in the case of flax) fragments in addition to respirable particles. When viewed with the light microscope, the fragments appeared as elongated, pointed structures. The possibility that certain of the fragments within corn, settled, and spring wheat were derived from cell walls was suggested by the detection of pentoses following colorimetric assay of neutralized 2 N trifluoroacetic acid hydrolyzates of these dusts. The presence of pentoses together with the occurrence of proteins within water washings of grain dusts suggests that glycoproteins may be present within the dusts. With scanning electron microscopy, each dust was found to consist of a distinct assortment of particles in addition to respirable particles. Small husk fragments and "trichome-like" objects were common to all but corn dust.

Elemental analysis of airborne grain dusts.

The elemental composition of a group of airborne and settled grain dusts is reported. This survey was undertaken as part of a study to systematically describe the chemistry and morphology of these representative dusts. Our data show that airborne or settled grain dusts differ from each other with respect to elemental composition. Such fundamental differences may be related to previously observed differences in the biological activities of the dusts.

Interactions of complement with an extract of airborne spring wheat dust.

The inhalation, by grain elevator workers, of airborne grain dusts can lead to pulmonary problems. Complement, which is present in human airways, can interact with various grain dusts, producing activation products that have been shown to participate in the inflammatory reaction. Because of this apparent connection between grain-dust inhalation, complement activation, and respiratory difficulties, we are studying the reaction of an aqueous extract of spring wheat dust (swd) with human complement. The swd extract activates both the classical and alternative pathways; it acts on purified C2 to inhibit it, and it reacts with undiluted serum to consume C4 with kinetics significantly different from those shown by a "typical" antigen-antibody complex (sensitized sheep erythrocytes). Enzyme susceptibility experiments suggest that the alternative and classical pathway activators of swd extract are neither protein nor nucleic acid; periodate oxidation indicates these substances are carbohydrate, and gel filtration suggests they are macromolecular. Enzyme vulnerability also indicates that the C2 inhibitor of swd extract is ribonucleic acid. Although endotoxin is present in swd extract, a gel-filtration experiment showed that a major fraction of the complement reactivity was not associated with this substance.

Reactivity of vitamin A derivatives and analogues with vitamin A antibodies.

Vitamin A antibodies were obtained using retinoic acid conjugated to human serum albumin as an immunogen. The following constraints governed the reactivity of vitamin A analogues with such an anti-serum. The stereochemistry of the side chain is relatively unimportant, and 9- and 13-cis retinal react almost as well as all-trans retinal. The nature of the ring is important; all of the compounds that react readily carry a beta-ionic ring; all of the compounds bearing an aromatic ring react poorly; the two compounds that display intermediate reactivity have non-aromatic 6- and 5-membered rings, respectively.

Terminal stages of complement hemolysis: technical comment.

In studying terminal lytic events of sensitized erythrocytes which have reacted with all 9 components of complement, we agree in general with the conclusions of Boyle and Borsos [1] that the cellular intermediate passes through the stages E*bound, E*inserted, and E*doomed. However, we differ with these workers on the influence of 3 variables on the terminal reactions. These variables are: time of hemolysis; temperature of E* preparation; and the use of EDTA to manipulate E*.

Large scale isolation of functionally active components of the human complement system.

In the present work a scheme is presented for the isolation of multiple components of human complement in a functionally and biochemically pure state and with full hemolytic activity. These preparative procedures allow high recovery of milligram and gram quantities of particular complement components from a large pool (2-11 liters) of fresh EDTA plasma in no more than four chromatographic steps. Many components (C3bINA, C5, C3, C1EI, C4, and C9) are recovered functionally pure or highly purified following the first chromatographic step employing DEAE-Sephacel and may be utilized as reagents with no further purification. Prior to anion exchange, individual units of plasma are treated with inhibitors of complement activation and serum proteases, the pooled plasma is fractionated with polyethylene glycol, depleted of plasminogen on Sepharose-lysine, and rapidly ultrafiltered to low ionic strength and high protein concentration. The high degree of resolution of the components on DEAE-Sephacel subsequently obtained is demonstrated by the functional recovery and purification in a representative experiment as indicated (in their order of elution) for the following proteins: C3bINA (24%, 18-fold), C2 (74%, 12-fold), C7 (87%, 14-fold), factor B (55%, 8.7-fold),, C8 (50%, 16-fold), C6 (82%, 25-fold), beta 1H (39%, 12-fold), C5 (62%, 111-fold), C3 (99%, 64-fold), C1EI (42%, 135-fold), C9 (80%, 297-fold), and c4 (78%, 164-fold). Other components separated by these procedures include C1q and C4 binding protein. Additional steps described, which demonstrate the utility and effectiveness of this preparative scheme, have allowed isolation of C3, C5, and C7 as pure components with full hemolytic activity as judged by functional, immunochemical, and physicochemical criteria. C8, also isolated as a homogeneous protein, was recovered with partial hemolytic activity. All these components were recovered in high yield and in the purification as indicated: C3 (61%, 103-fold), C5 (24% 1350-fold), C7 (19%, 2260-fold), and C8 (32%, 547-fold). Complement components C6, beta 1H, factor B, and C2 in addition to C3bINA, C1EI, C4, and C9 are recovered partially purified with good activity and are amenable to further purification.

Vitamin A antibodies: application to radioimmunoassay.

A radioimmunoassay for serum vitamin A is described which can detect as little as 1 ng of retinol. The statistical characteristics of this assay are presented and its use in a nutritional experiment is discussed.

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.