Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m?g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.     

The Changing Alaskan Experience—Health Care Services and Cultural Identity

Before Western contact, Alaskan Native populations were self-sufficient in their health practices. Slowly, the Native health care system was replaced by a Western one which was highly effective in treating infectious diseases. As infectious diseases were brought under control by the Indian Health Service, the emergent leading health problems were related to violence, attributed in part to cultural disintegration. New types of Native health providers and new Native-controlled institutions evolved to provide culturally appropriate health and mental health services and to promote a stronger cultural identity.     

Highly Purified pp60src Induces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.     

Extrachromosomal Elements in Group N Streptococci

The deoxyribonucleic acid (DNA) of Streptococcus lactis C2, S. cremoris B(1), and S. diacetilactis 18-16 was labeled by growing cells in Trypticase soy broth containing (3)H-labeled thymine. The cells were gently lysed with lysozyme, ethylenediaminetetraacetic acid, and sodium lauryl sulfate. The chromosomal DNA was separated from plasmid DNA by precipitation with 1.0 M sodium chloride. The existence of covalently closed circular DNA in the three organisms was shown by cesium chloride-ethidium bromide equilibrium density gradient centrifugation of the cleared lysate material. In an attempt to correlate the loss of lactose metabolism with the loss of plasmid DNA, lactose-negative mutants of these organisms were examined for the presence of extrachromosomal particles. Covalently closed circular DNA was detected in the lactose-negative mutants of S. lactis C2 and S. diacetilactis 18-16. In S. cremoris B(1), however, no covalently closed circular DNA was observed by using cesium chloride-ethidium bromide gradients. Electron micrographs of the satellite band material from S. lactis C2 and its lactose-negative mutant confirmed the presence of plasmid DNA. Three distinct plasmids having approximate molecular weights of 1.3 x 10(6), 2.1 x 10(6), and 5.1 x 10(6) were observed in both organisms.     

Differentiation studies on separated rabbit early erythroid cells

Haemoglobin-containing cells were removed from cell suspensions of adult rabbit bone marrow by immune lysis, and the remaining cells were layered into BSA density gradients. The top fractions contained early erythroid cells, while fractions near the bottom of the gradient contained granulocytes. Two populations of erythroid cells from anaemic rabbits were resolved by the gradient which differed in their time of maximum stimulation of haem synthesis, in culture with erythropoietin. In addition, a difference in requirement for the presence of erythropoietin in the culture medium was found in separated erythroid cells from rabbits with varying degrees of anaemia.     

Cytoplasmic and nuclear inheritance of resistance to alkylguanidines and ethidium bromide in a petite-negative yeast

Mutants of the petite-negative yeast, $\text{Kluyveromyces}$$\text{lactis}$, resistant to the inhibitors of oxidative phosphorylation, decamethylenediguanidine and octylguanidine were isolated from medium containing ethidium bromide. All mutants were resistant to ethidium bromide; some mutants were resistant to both alkylguanidines, some to one, others to neither. Both nuclear and cytoplasmic inheritance of resistance to decamethylenediguanidine and ethidium bromide was demonstrated by tetrad analysis.     

Capsella embryogenesis: The suspensor and the basal cell

Summary The suspensor and basal cell ofCapsella were examined with the electron microscope and analyzed by histochemical procedures. The suspensor cells are more vacuolate and contain more ER and dictyosomes, but fewer ribosomes and stain less intensely for protein and nucleic acids than the cells of the embryo. The end walls of the suspensor cells contain numerous plasmodesmata but there are no plasmodesmata in the walls separating the suspensor from the embryo sac. The lower suspensor cells fuse with the embryo sac wall and the lateral walls of the lower and middle suspensor cells produce finger-like projections into the endosperm. At the heart stage the suspensor cells begin to degenerate and gradually lose their ability to stain for protein and nucleic acids.The basal cell is highly vacuolate and enlarges to a size of 150 X 70. An extensive network of wall projections develops on the micropylar end wall and adjacent lateral wall. The nucleus becomes deeply lobed and suspended in a strand of cytoplasm traversing the large vacuole. The cytoplasmic matrix darkens at the late globular stage and histochemical staining for protein becomes very intense. The basal cell remains active after the suspensor cytoplasm has degenerated. It is proposed that the suspensor and basal cell function as an embryonic root in the absorption and translocation of nutriments from the integuments to the developing embryo.Research supported by NSF grant GB 3460 and NIH grant 5-RO 1-CA-03656-09.