The organization of synaptic axcplasm in the lamprey (petromyzon marinus) central nervous system.

The fine structure of synapses in the central nervous system of lamprey (Petromyzon marinus) ammocoetes has been investigated. Both synapses within the neuropil and synaptic links between giant fibers (including Müller cells) and small postsynaptic units are described. The distribution of neurofilaments and microtubules in nerve profiles over a wide diameter range is described, and the possible role of these structures in intracellular transport is discussed. Electron micrographs indicate that small lucent "synaptic vesicles" occur sparsely throughout the axoplasm and in regular arrays in association with microtubules in the vicinity of synapses. Within a synaptic focus, immediately adjoining the presynaptic membrane, vesicles are randomly arranged and are not associated with microtubules. Neurofilaments are present, generally in large numbers, but these are not associated with vesicles or other particulates. The structural findings are considered in terms of current concepts of fast and slow transport in neurons and the mechanochemical control of intracellular movement of materials.

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.

Subcellular localization studies indicate that lipoxygenases 1 to 6 are not involved in lipid mobilization during soybean germination

Soybean (Glycine max) lipoxygenase (LOX) has been proposed to be involved in reserve lipid mobilization during germination. Here, subcellular fractionation studies show that LOX1, -2, -3, -4, -5, and -6 isozymes were associated with the soluble fraction but not with purified oil bodies. The purified oil bodies contained small amounts of LOX1 (<0.01% total activity), which apparently is an artifact of the purification process. Immunogold labeling indicated that, in cotyledon parenchyma cells of LOX wild-type seeds that had soaked and germinated for 4 d, the majority of LOX protein was present in the cytoplasm. In 4-d-germinated cotyledons of a LOX1/2/3 triple null mutant (L0), a small amount of label was found in the cytoplasm. In epidermal cells, LOX appeared in vacuoles of both wild-type and L0 germinated seeds. No LOXs cross-reacting with seed LOX antibodies were found to be associated with the cell wall, plasma membrane, oil bodies, or mitochondria. Lipid analysis showed that degradation rates of total lipids and triacylglycerols between the wild type and L0 were not significantly different. These results suggest that LOX1, -2, -3, -4, -5, and -6 are not directly involved in reserve lipid mobilization during soybean germination.

Unusual Cytoplasmic Inclusions Induced in Tobacco by Peanut Stunt Virus Subgroup II Strains Map to RNA3.

ABSTRACT Infection of tobacco protoplasts or leaf tissues with peanut stunt virus (PSV) subgroup II strains induced the production of unusual cytoplasmic ribbon-like inclusions. The inclusion structures appeared as long, thin, densely staining sheets that were prevalent within the cytoplasm, accumulating most commonly near vacuoles. Numerous virions and ribosomes could be seen adjacent to the inclusion surfaces. The formation of these novel inclusions appeared to be subgroup specific, since infection of tobacco with PSV strains W and B (subgroup II), but not strains ER, V, and J (subgroup I), induced the inclusions. Furthermore, inclusion formation was shown to be host specific, because the inclusions were not detected in either of two leguminous host species infected with PSV subgroup II strains. Using tobacco protoplasts electroporated with various assortments of infectious RNA transcripts derived from cDNA clones of genomic RNAs of PSV-ER and PSV-W, we demonstrated that induction of the unusual ribbon-like inclusions maps to PSV-W (subgroup II) RNA3. This conclusion is consistent with the finding that PSV strain BV-15, a natural intraspecific reassortant that derives its RNA2 and RNA3 from a subgroup I strain, did not induce inclusion formation.

Expression of the Peanut stunt virus Coat Protein Gene Is Essential and Sufficient for Production of Host-Dependent Ribbon-Like Inclusions in Infected Plants.

ABSTRACT We previously have reported that infection of tobacco protoplasts or leaf tissue with the cucumovirus Peanut stunt virus (PSV) induced the production of unusual cytoplasmic ribbon-like inclusions. The formation of these novel inclusions is strain-specific, because infection of tobacco with subgroup II PSV strains, but not subgroup I strains, induced the production of inclusions. Furthermore, we have demonstrated that induction of the ribbon-like inclusions maps to PSV subgroup II RNA3, which codes for the coat protein (CP) and movement protein (MP). We have now extended these studies using chimeric constructs containing CP and MP open reading frames (ORFs) from PSV strains ER and W that belong to subgroups I and II, respectively. Additionally, recombinant Potato virus X (PVX) vectors containing translatable and untranslatable PSV CP ORF were constructed. Plants inoculated with infectious chimeric PSV or recombinant PVX transcripts were analyzed for CP expression by enzymelinked immunosorbent assay and reverse transcription-polymerase chain reaction and for inclusion production by electron microscopy. The results of these experiments indicated that translation of the CP ORF alone is essential and sufficient for inclusion production. In immunogold labeling experiments using an antiserum to PSV virions, abundant gold labeling of the inclusions was observed, suggesting that PSV CP is probably a major component of the inclusions. Because inclusion production is host specific, a host factor is likely to be involved. In addition to their diagnostic importance, these novel inclusions may also prove valuable in identifying the host factors that interact with PSV CP.

The fine structure of muscle attachments in a spider (Latrodectus mactans, Fabr.).

The fine structure of a spider myo-apodeme junction is described, and discussed in terms of other arthropod muscle attachments. This is contrasted with the situation in the venom gland, equipped with muscle fibers that control expulsion of the secreted material. The latter involves a cell-free collagenous matrix, lying between the muscle cells and the sheath of the gland. As in other arthropods, skeletal fibers are attached to the apodeme cuticle via specialized epidermal cells, containing oriented microtubules. Interdigitations between these cells and muscle, basally, and cuticle, apically, are described. Extracellular tonofibrillae described elsewhere are inconspicuous in the apodeme cuticle.

Purification of monolayer cell cultures of the endocrine pancreas.

Experimental use of primary cultures of endocrine pancreas is constrained by early, vigorous proliferation of fibroblastoid cells. The addition of heavy metals, sodium ethylmercurithiosalicylate, phenyl mercuric acetate, phenyl mercuric nitrate and sodium aurothiomalate to the culture media selectively destroys these fibroblastoid cells yielding highly enriched, morphologically intact, functionally competent endocrine cells that are capable of cell replication. This action of heavy metals appears to be due to reversible inhibition of sulfhydryl enzymes since glutathione and thioglycolate were demonstrated to completely inhibit the cytotoxic effects of the mercury and gold containing agents, respectively. Certain variables in the application of the mercurial agents to pancreatic endocrine cell cultures were defined, most notably the enhanced sensitivity of fetal vs. neonatal tissue, and in inverse relationship of cell density to effective toxicity. After removal of the heavy metal agent from the culture media, many pancreatic islets send out cytoplasmic projections, containing large numbers of oriented microtubules which serve as bridging units to adjacent endocrine cells. The sustained availability of virtually pure pancreatic endocrine cell cultures, which results from the application of mercury to the culture media will undoubtedly permit many aspects of the cell biology of the endocrine pancreas to be directly and sequentially assailed.

Structural modulations in the tsetse fly milk gland during a pregnancy cycle.

Gross ultrastructural and histochemical details of the integumental milk glands of the tsetse fly Glossina morsitans have been examined during the pregnancy cycle. Structural evidence for protein secretion is found between Days 3-8 of the nine-day cycle: termination of activity is completed on the day of parturition. Onset of lactation is synchronized with the eclosion of the first instar larva. The changes in cell volume (notably in the extracellular reservoir) occurring throughout the pregnancy cycle are illustrated in electron micrographs, and a one hundred-fold volume increase in the reservoir volume between the inactive phase and the active period is illustrated and discussed in terms of membrane modulation of the limiting membrane of the reservoir. Intracellular membrane changes during the cycle, particularly the development of extensive ER arrays in the actively secreting cell, are illustrated and discussed. It is suggested that cytoplasmic microtubules play a part in maintaining the form of the distended secretory cell, at the height of secretory release and storage. Histochemical observations on the milk secretion, and the contents of the larval gut are presented.

Structural cross-bridges between microtubules and mitochondria in central axons of an insect (Periplaneta americana).

The distribution of microtubules and mitochondria in central axons of an insect (Periplaneta americana) is assessed by comparison between counts on micrographs and computed axon random 'models'. These studies show that the observed multiple association of microtubules with individual mitochondria is statistically highly significant. Electron micrographs of thin sections show that linkage is effected by physical cross-bridge, possibly comprising components from the microtubule and mitochondrion. Linear particle arrays are described on the outer mitochondrial membrane in freeze-fracture replicas, and tentatively related to the bridges seen in thin sections. The results are discussed in terms of proposed roles of microtubules in neurons and other cells.

Effects of Microplitis croceipes Teratocytes on Host Haemolymph Protein Content and Fat Body Proliferation.

Qualitative and quantitative changes in haemolymph proteins in Heliothis virescens were observed in larvae injected with either Microplitis croceipes teratocytes or teratocyte secreted proteins (TSP). Haemolymph protein titres in hosts receiving either 0.5 or 1 larval equivalent (LE) of teratocytes were similar to those of parasitized larvae, whereas a single injection of 4LE of TSP was required to induce a similar response. SDS-PAGE showed that the 82kDa monomer of riboflavin-binding protein and the 74/76kDa monomers of storage proteins were significantly reduced in parasitized larvae and in nonparasitized larvae treated with TSP. Concentrations of a 155kDa monomer (insectacyanin chromoprotein) also were reduced in parasitized larvae and those injected with either teratocytes or TSP. Two monomers (56 and 60kDa) were unique to parasitized larvae. Treated larvae required several days longer than controls to reach a comparable premetamorphic stage (burrowing-digging). Reductions in fat body proliferation similar to those seen in parasitized larvae were observed in larvae treated with either 1LE of teratocytes, or with 2 or 4LEs of TSP. Perivisceral fat body weights from larvae treated with either 0.25 or 0.5LE of teratocytes were significantly reduced, but less so than those which received 1LE. Thus, fat body proliferation in both teratocyte- and TSP-treated larvae was inhibited in a dose-dependent manner. Both light- and transmission electron microscopy observations revealed cytological differences in fat body tissues of larvae injected with either teratocytes or TSP from the condition observed in parasitized larvae and noninjected controls. Gross dissection of periviseral fat body from parasitized, teratocyte-injected and TSP-injected larvae showed tissue much less developed and differing considerably in appearance from controls. Observed differences included reduced size and/or number of lipid bodies and qualitative and quantitative changes in other cytoplasmic organelles.