The interactions of bilirubin with model and biological membranes

The partitioning of bilirubin between albumin and model and biological membranes and the differential partitioning of bilirubin between membranes with different lipid and protein compositions were measured. Partition coefficients were independent of the concentration of bilirubin in membranes up to at least 7 mol of bilirubin/mol of phospholipid. The avidity of albumin for bilirubin was greater than that of membranes, but the avidity of the latter for bilirubin depended on the composition of the membrane. Bilirubin partitioned preferentially into model membranes comprised of microsomal lipids greater than dioleoylphosphatidylcholine = plasma membrane lipids much greater than egg phosphatidylcholine = dimyristoylphosphatidylcholine. Partitioning into membranes was increased if these contained proteins, but the effect of proteins could not be attributed to specific binding to sites on proteins, as reflected by the temperature independence of partition coefficients. Differential partitioning of bilirubin into different membranes of pure lipids also was independent of temperature. Differences in the bulk phase fluidity of membranes does not appear to account for the preferential partitioning of bilirubin into some membranes. It appears that bilirubin partitions into elements of free volume of differing sizes in membranes with variable lipid compositions and that the size of these elements can be increased by adding proteins to membranes.     

A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.     

Thermal denaturation and loss of viability in Escherichia coli and Bacillus stearothermophilus

When Escherichia coli was heated at 10°C/min in a differential scanning calorimeter, the onset of irreversible thermal denaturation occurred at 51°C, about 5°C above the maximum growth temperature. The temperature at which death rate was maximal (63°C) coincided with the thermogram peak caused by denaturation of the 30S ribosomal subunit. The maximum death rate in vegetative cells of Bacillus stearothermophilus occurred at the higher temperature of 71°C which also coincided with the leading edge of the main thermogram peak.     

Stabilization of proteins immobilized on Sepharose from leakage by glutaraldehyde crosslinking

A technique has been developed to prevent leakage of proteins immobilized on Sepharose without destroying their biological functions. This involves the use of glutaraldehyde at concentrations ranging from 0.015 to 0.25% ($\text{v}\text{v}$) to crosslink proteins, which had been coupled to Sepharose by conventional methods. Glutaraldehyde crosslinking decreases immuno-globulin G leakage from Sepharose-immunoadsorbents to undetectable levels without noticeably affecting antigen-binding activity and reduces leakage of lactoperoxidase from solid-phase lactoperoxidase with only a moderate reduction of enzymatic activity.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Sequence-based genetic markers for genes and gene families: single-strand conformational polymorphisms for the fatty acid synthesis genes of Cuphea

 Gene sequences are rapidly accumulating for many commercially and scientifically important plants. These resources create the basis for developing sequence-based markers for mapping and tracking known (candidate) genes, thereby increasing the utility of genetic maps. Members of most of the gene families underlying the synthesis of seed oil fatty acids have been cloned from the medium-chain oilseed Cuphea. Allele-specific-PCR (AS-PCR) and single-strand conformational polymorphism (SSCP) markers were developed for 22 fatty acid synthesis genes belonging to seven gene families of Cuphea using homologous and heterologous DNA sequences. Markers were developed for 4 fatty-acyl-acyl carrier protein thioesterase, 2 β-ketoacyl-acyl carrier protein synthase I, 4 β-ketoacyl-acyl carrier protein synthase II, 3 β-ketoacyl-acyl carrier protein synthase III, 3 acyl carrier protein, 2 β-ketoacyl-acyl carrier protein reductase, and 4 enoyl-acyl carrier protein reductase loci. Eighty-eight percent (14 of 16) of the SSCP loci were polymorphic, whereas only 9% (2 of 22) of the AS-PCR loci were polymorphic. These markers were mapped using a Cuphea viscosissima×C. lanceolata F₂ population and produced linkage groups of 10, 3, and 2 loci (3 loci segregated independently). The 10-locus linkage group had every gene but one necessary for the synthesis of 2- to 16-carbon fatty acids from acetyl-CoA and malonyl-ACP (the missing gene family was not mapped). SSCP analysis has broad utility for DNA fingerprinting and mapping genes and gene families. Received: 3 May 1996 / Accepted: 30 August 1996     

Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation

The interleukin-21 (IL-21)-IL-21-receptor system was discovered in 2000. It was immediately of great interest because of the homology of IL-21 to IL-2, IL-4 and IL-15, and of the IL-21-receptor subunit IL-21R to the beta-subunit of the IL-2 receptor, and because the IL-21 receptor also contains the common cytokine-receptor gamma-chain, the protein that is mutated in X-linked severe combined immunodeficiency. As we discuss, IL-21 has pleiotropic actions, from augmenting the proliferation of T cells and driving the differentiation of B cells into memory cells and terminally differentiated plasma cells to augmenting the activity of natural killer cells. Moreover, it has antitumour activity and might have a role in the development of autoimmunity, so these findings have implications for the treatment of cancer and autoimmune diseases.     

D2O induced alterations of mitosis in PtK1 cells

Deuterium oxide (D2O) was applied to PtK1 cells to assess its effect on mammalian mitosis. Cells exposed to culture medium containing up to 50% D2O were able to enter and complete mitosis, but the duration of mitosis was increased proportionally to the concentration of D2O applied. Cells exposed to 50% D2O showed increases of more than 300% for the interval between nuclear envelope breakdown and anaphase onset, and approximately 65% for the interval between anaphase onset and initial furrowing. At a concentration of 80%, D2O acted as an inhibitor of mitosis; after 8 h exposure to this concentration, cultures showed an increase in the proportion of mulinucleate cells and an absence of mitotic figures. When applied early in anaphase, 80% D2O effectively slowed chromosome separation, prolonging anaphase for more than 60 min. Normal chromosome motion was restored when medium containing D2O was replaced with control medium. Mitotic chromosomes remained condensed throughout prolonged anaphase intervals. Immunofluoresence examination of spindles stained using a monoclonal anti-tubulin revealed no pronounced increase in microtubule polymerization after exposure of cells to 20-80% D2O.