Crosslinking of phycobiliproteins from the cyanobacterium Mastigocladus laminosus with bis-imidates: localization of an intrasubunit and an intersubunit crosslink in C-phycocyanin

The light-harvesting pigment-protein complexes allophycocyanin (AP), C-phycocyanin (PC) and phycoerythrocyanin (PEC) of the cyanobacterium Mastigocladus laminosus consist of alpha- and beta-subunits containing about 170 amino-acid residues each. These two subunits form an alpha,beta-monomer, three of which build up a disc-shaped trimer. In this study these phycobiliproteins were crosslinked with bis-imidates. Various spacer lengths of the reagent and various aggregation states of the phycobiliprotein were tested. An intersubunit crosslink could be verified in all three phycobiliproteins. PC-trimers were crosslinked with the homobifunctional reagent dimethyl pimelimidate having a maximal crosslinking distance of 10 A. Two crosslinks could be identified: an intramonomer intersubunit crosslink with a yield of 48% and an intrasubunit crosslink within alpha PC (57%). These products were chemically and enzymatically fragmented and the small crosslinked peptides were isolated and then identified by amino-acid analysis. The following amino acids were crosslinked: alpha-Val 1 with beta-Ala 1 and alpha-Lys 62 with alpha-Lys 134. Both crosslinks could be localized within the known three-dimensional structure of PC.     

Heterogeneity of intercellular adhesion in rat liver cells in culture

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.     

Forces in the spider orb web

Summary Pretensile forces were measured in individual threads of intact spider webs. In the orb web of Araneus diadematus forces decrease from mooring threads to frame threads and radii, a typical ratio being 1071. The smaller number of radii in the upper than in the lower half of the orb is paralleled by force ratios of 21 to 31. A similar difference between radii built first during web construction and radii added after completion of the frame underlines the importance of the former as part of the scaffolding. High tensions in the auxiliary spiral stabilize the radii in addition to providing a pathway for the spider when inserting the sticky spiral. Radial pretension (F) changes with spider mass (m). F/m is similar for different animals indicating an adaptation of radial forces to those resulting from spider mass. Several observations suggest tension control by the spider. When forced to anchor its web to thin flexible rods tension in the threads remains in the normal range. Tension values are similar in the webs of A. diadematus, Zygiella x-notata, Nuctenea umbratica, and Nephila clavipes indicating independence from details of web geometry. Only the mooring threads of Nephila show unusually large forces suggesting a narrower working range of tensions for the catching area than for the scaffolding.     

The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region.

PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3. Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene. In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3. Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs. The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.     

Transformation of bacteria with plasmid DNA by electroporation

The possibility of electric field-mediated transformation ("electroporation") of a gram-positive bacterium (Enterococcus faecalis) and two gram-negative bacteria (Escherichia coli and Pseudomonas putida) with plasmid DNA was investigated. E. faecalis protoplasts could be transformed by electroporation with a transformation frequency of 10(4) to 10(5) transformants/micrograms plasmid. Untreated--i.e., washed--cells of E. coli could be transformed with rates of 1 X 10(5) transformants/micrograms plasmid DNA. Transformation rates for P. putida cells were up to 3 X 10(4) if the method developed for E. coli was used. Detailed protocols for these systems, including the results of various optimization experiments, are given.     

Structure and genomic organization of a new family of murine retrovirus-related DNA sequences (MuRRS).

A new class of murine retrovirus-related sequences (MuRRS) is described. These 5.7 kb long transposon-like DNA-elements start and end with approximately 600 bp long repeats identical to previously identified solitary LTR-like elements (LTR-IS). There are about 50 - 100 5.7 kb elements and about 500 - 1000 solo LTR-IS elements per mouse haploid genome. Sequence analysis of one cloned MuRRS element revealed several possible open reading frames with partial sequence homologies to retroviral gag, pol and env genes.     

Macrophage mediation of the inhibitory effects of elevated intracellular levels of adenosine-3':5' cyclic monophosphate (cAMP) on macrophage-Trypanosoma cruzi association

Presence of theophylline and dibutyryl-cAMP—agents which cause elevation of intracellular levels of cAMP—in in vitro systems in which murine macrophages interact with virulent blood forms of Trypanosoma cruzi resulted in a marked inhibition of cell-parasite association (i.e., decreased surface binding and/or internalization). This effect was evidenced in terms of significant reductions in both the percentage of infected macrophages and the average number of trypanosomes per 100 macrophages. Pretreatment of the macrophages with these agents produced a similar inhibition whereas pretreatment of the parasites had no significant consequence on the interaction. The inhibitory effect was transient since it was no longer seen after 30 min of incubation of the treated macrophages in fresh medium. Thus, the inhibitory effect is exerted through a transient effect on the macrophage.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.