Peptide crystal growth via sulfonate salt formation: The structure of bis-glycylglycine-1,5-naphthalenedisulfonate hydrate

Summary Continuing a line of investigations on methods for formation and growth of high-quality crystals of peptides, the glycylglycine sequence has been crystallized by evaporation methods as a salt with 1,5-naphthalenedisulfonic acid. The structure of the peptide is highly extended, and is conformationally quite similar to the structures which have been characterized for other zwitterionic and salt forms of this sequence. Thus, crystallization as a salt with this sulfonic acid has imposed no undue influence upon the molecular conformation. These results offer further indication that the preparation of peptide sulfonate salts, particularly with arene templates, may have broad general utility for crystallization of interesting sequences which until now have not been approachable in their zwitterionic forms.     

BRCA1 maps proximal to D17S579 on chromosome 17q21 by genetic analysis

Previous studies have demonstrated linkage between early-onset breast cancer and ovarian cancer and genetic markers on chromosome 17q21. These markers define the location of a gene (BRCA1) which appears to be inherited as an autosomal dominant susceptibility allele. We analyzed five families with multiple affected individuals for evidence of linkage to the BRCA1 region. Two of the five families appear to be linked to BRCA1. One apparently linked family contains critical recombinants, suggesting that the gene is proximal to the marker D17S579 (Mfd188). These findings are consistent with the maximum-likelihood position estimated by the Breast Cancer Linkage Consortium and with recombination events detected in other linked families. Linkage analysis was greatly aided by PCR-based analysis of paraffin-embedded normal breast tissue from deceased family members, demonstrating the feasibility and importance of this approach. One of the two families with evidence of linkage between breast cancer and genetic markers flanking BRCA1 represents the first such family of African-American descent to be reported in detail.     

Ecological distinctions between sympatric species of Saguinus and Sciurus

Tamarins are small New World monkeys that have been described as "squirrellike." Squirrels, along with bats and birds, are the taxa most likely to utilize resources similar to those used by primates in the tropical forest canopy. In this paper we compare differences in ecology, diet, locomotion, and habitat utilization between sympatric populations of tamarins (Saguinus oedipus) and tree squirrels (Sciurus granatensis) in Panama. Data presented indicate that although there is some degree of resource overlap, patterns of habitat utilization differ significantly. Rather than being "squirrellike," the Panamanian tamarin exhibits a pattern of locomotor and feeding behavior consistent with that found in other arboreal primates.     

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity

We have suggested previously that the amino-terminal 8 kilodaltons of pp60^src may serve as a structural hydrophobic domain through which pp60^src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60^src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, in contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60^src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60^src, and suggest that the amino terminus of pp60^src may have a critical role in the promotion of in vivo tumorigenicity.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Effect of Light and Chilling Temperatures on Chilling-sensitive and Chilling-resistant Plants. Pretreatment of Cucumber and Spinach Thylakoids in Vivo and in Vitro

The effects of chilling temperatures, in light or dark, on the isolated thylakoids and leaf discs of cucumber (Cucumis sativa L. “Marketer”) and spinach (Spinacia oleracea L. “Bloomsdale”) were studied. The pretreatment of isolated thylakoids and leaf discs at 4 C in the dark did not affect the phenazine methosulfate-dependent phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, or chlorophyll content. Exposure of cucumber cotyledon discs and isolated thylakoids of cucumber and spinach to 4 C in light resulted in a rapid inactivation of the thylakoids. The sequence of activities or components lost during inactivation (starting with the most sensitive) are: phenazine methosulfate-dependent cyclic phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, and chlorophyll. The rate of loss of proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity and chlorophyll is similar for isolated cucumber and spinach thylakoids, whereas spinach thylakoids are more resistant to the loss of phenazine methosulfate-dependent phosphorylation. The thylakoids of spinach leaf discs were unaffected by exposure to 4 C in light. The results question whether the extreme resistance of spinach thylakoids treated in vivo is solely a function of the chloroplast thylakoid membranes and establish the validity of using in vitro results to make inferences about cucumber thylakoids treated in vivo at 4 C in light.     

Influence of Soil Fumigation on the Fusarium-Root-knot Nematode Disease Complex of Cotton in California

For control of the root-knot nematode, Meloidogyne incognita, and the pathogenic wilt fungus, Fusarium oxysporum, on cotton, soil fumigants were applied in the field at conventional and higher rates. Conventional rates suppressed Fusarium wilt but higher rates gave quicker early growth, better stands, less stand loss over the season, a lower percentage of plants infected with wilt, fewer plants with vascular discoloration, and fewer nematodes. The best treatment about doubled the yields of untreated controls in one experiment and quadrupled them in another.     

Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications.

A complete protocol for the expression of recombinant cytochrome c genes from yeast, Drosophila melanogaster, and rat in a yeast strain, GM-3C-2, which does not express its own cytochromes c is described. The construction of the expression vectors, transformation and large-scale growth of the yeast, and preparation and purification of the recombinant cytochromes c are described. It was found that, contrary to the way yeast modifies its own cytochromes c, the recombinant proteins were partially acetylated at their N-terminus, except for the drosophila protein, which remained entirely unblocked. Furthermore, the yeast and rat proteins were close to fully trimethylated at lysine 72, while the drosophila protein could be separated chromatographically into forms containing tri-, di-, mono-, and unmethylated lysine 72 showing corresponding resonances in the NMR spectrum. These observations emphasize that, in employing expression procedures to obtain native or mutant forms of cytochrome c, it is essential to identify the variety and extent of post-translational modifications and to separate the preparation into pure monomolecular species. Otherwise, it may become impossible to distinguish between the influence of a site-directed mutation and unexamined post-translational modifications.