Comparison of the primary structures of clathrin light chains from bovine brain and adrenal gland by peptide mapping

The structures of the polymorphic forms of clathrin light chains were analyzed by two peptide mapping procedures. Comparison of the products of partial digestion by V8 protease showed no common peptides between LCA and LCB from bovine brain. No similarities between clathrin light chains and tropomyosin chains from bovine brain and skeletal muscle were detected with this technique. The peptides produced by complete tryptic digestion of LCA and LCB from bovine brain and bovine adrenal gland were analyzed by reverse phase h.p.l.c. For both LCA and LCB the polypeptides from different tissues showed considerable homology. LCA from brain and adrenal gland shared 10 out of a total of 15 peptides. LCB from brain and adrenal gland shared 10 out of 14 peptides. In contrast, when LCA was compared with the LCB chain from the same tissue very few peptides were shared; 4/23 for brain and 3/21 for adrenal gland. These results strongly indicate that, within a tissue, LCB is not related to LCA by post-translational processing and that each chain is encoded by a separate gene. The data also demonstrate the close homology of the different forms of LCA and LCB expressed in different tissues within the same organism. Thus the polymorphic differences of clathrin light chains within a tissue are greater than those between tissues.     

Sec16 influences transitional ER sites by regulating rather than organizing COPII

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.     

The roles and values of wild foods in agricultural systems

Almost every ecosystem has been amended so that plants and animals can be used as food, fibre, fodder, medicines, traps and weapons. Historically, wild plants and animals were sole dietary components for hunter–gatherer and forager cultures. Today, they remain key to many agricultural communities. The mean use of wild foods by agricultural and forager communities in 22 countries of Asia and Africa (36 studies) is 90–100 species per location. Aggregate country estimates can reach 300–800 species (e.g. India, Ethiopia, Kenya). The mean use of wild species is 120 per community for indigenous communities in both industrialized and developing countries. Many of these wild foods are actively managed, suggesting there is a false dichotomy around ideas of the agricultural and the wild: hunter–gatherers and foragers farm and manage their environments, and cultivators use many wild plants and animals. Yet, provision of and access to these sources of food may be declining as natural habitats come under increasing pressure from development, conservation-exclusions and agricultural expansion. Despite their value, wild foods are excluded from official statistics on economic values of natural resources. It is clear that wild plants and animals continue to form a significant proportion of the global food basket, and while a variety of social and ecological drivers are acting to reduce wild food use, their importance may be set to grow as pressures on agricultural productivity increase.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

Cyclic amp-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus: Expression of transformation-associated markers

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by α-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.