Effects of phospholipases,proteases and neuraminidase on γ-hydroxybutyrate binding sites

Summary -Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A₂ and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.Abbreviations GHB -hydroxybutyrate - LPC L--lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - BSA Bovine Serum Albumin     

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.     

The QM gene is X-linked and therefore not involved in suppression of tumorigenesis in Wilms' tumor

Summary Inactivation of one or more tumor-suppressor genes on the short arm of chromosome 11 is thought to play a role in the etiology of Wilms' tumor. A candidate gene, QM, was recently isolated by subtractive hybridization between a tumorigenic cell line (deleted for part of 11p) and a non-tumorigenic cell line (the tumorigenic cell line carrying an extra t(X;11)copy). We show here with an exon-specific polymerase chain reaction that the genomic homolog of the QM cDNA is located in the G6PD-color vision genes region in Xq28. No homologous sequences could be detected on 11p. Our experiments indicate that the QM gene is not involved in the suppression of Wilms' tumor.     

Polyomavirus large T mutants affected in retinoblastoma protein binding are defective in immortalization.

To clarify the relationship between the various activities of the polyomavirus large T antigen and the contribution of this oncogene to neoplastic transformation, we constructed a series of mutants with small deletions or single-amino-acid substitutions in two separate regions of the protein. These sequences were targeted because they showed considerable similarity to conserved regions 1 and 2 of adenovirus E1A which are thought to be binding sites for the retinoblastoma gene product (pRB). The pRB-binding properties of the large T mutants were assessed with an in vitro coimmunoprecipitation assay. pRB binding was readily detected with wild-type large T, but coprecipitation was completely abolished by as little as a single amino acid substitution (Asp-141----Glu or Glu-146----Asp) in region 2 of the polyomavirus large T antigen. Mutants defective in pRB binding were unable to immortalize primary rat embryo fibroblasts, suggesting that association with pRB is an important component of immortalization mediated by polyomavirus large T. The mutations in region 1 affected pRB binding only marginally, yet some of them severely impaired immortalization, indicating that pRB binding may be essential but not sufficient for immortalization.     

Anther-specific,developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower

We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

Techniques for encapsulating bioactive agents into liposomes

As a prerequisite for the use of liposomes for delivery of biologically active agents, techniques are required for the efficient and rapid entrapment of such agents in liposomes. Here we review the variety of procedures available for trapping hydrophilic and hydrophobic compounds. Considerations which are addressed include factors influencing the choice of a particular liposomal system and techniques for the passive entrapment of drugs in multilamellar vesicles and unilamellar vesicles. Attention is also paid to active trapping procedures relying on the presence of (negatively) charged lipid or transmembrane ion gradients. Such gradients are particularly useful for concentrating lipophilic cationic drugs inside liposomes, allowing trapping efficiencies approaching 100%.     

Ability of a T-antigen transport-defective mutant of simian virus 40 to immortalize primary cells and to complement polyomavirus middle T in tumorigenesis.

The oncogenic potential of polyomavirus in newborn rats could not be expressed by a genome encoding only the middle T antigen but required the presence of one of the other two viral early genes, small T or large T. The tumorigenicity defect could also be complemented by other viral or cellular genes that are known to be implicated in immortalization and establishment functions. The simian virus 40(cT)-3 mutant (R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984), which fails to localize to the nucleus, has the capacity to complement polyomavirus middle T in tumorigenesis and to immortalize primary rat embryo fibroblasts when it was cotransfected in the presence of pSV2-neo. Our data suggested that under the conditions of DNA-mediated tumor induction and cotransfection with a dominant selection marker, the cellular alterations achieved by nonnuclear oncogenes such as polyomavirus small T and simian virus 40(cT)-3 were sufficient to complement polyomavirus middle T in transformation and tumorigenesis.     

Apolipoprotein D and cross-reacting human plasma apolipoproteins identified using monoclonal antibodies

We have produced five hybridomas which secreted monoclonal antibodies that reacted with human plasma apolipoprotein D. On analysis by polyacrylamide gel electrophoresis (PAGE) high density lipoproteins and lecithin:cholesterol acyltransferase (EC 2.3.1.43)-enriched fractions of plasma contained many protein bands that reacted with the antibodies. Purified apolipoprotein D had the lowest Mr (29,000), the lowest pI (4.8-5.2), and the greatest migration on alkaline urea-PAGE of all the immunoreactive bands. These characteristics agreed with those described for apolipoprotein D in the literature. The other immunoreactive proteins had apparent Mr from about 39,000 to 98,000, they migrated more slowly than apolipoprotein D on alkaline urea-PAGE, and there were 10 polymorphs on isoelectric focusing. These cross-reacting proteins were present in the high density lipoproteins of each of four individuals sampled on several occasions and in pooled plasma. All of the monoclonal antibodies reacted both with apo-D and the higher Mr cross-reacting proteins. Each of our five monoclonal antibodies bound to one of two distinct antigenic sites on apo-D, determined by antibody competition immunoassays. Neither of these two sites was composed of carbohydrate, but expression of both sites seemed to be influenced by thiol-reducing agents: site 5G10 gained but 4E11 either lost immunoreactivity or was unchanged by reduction according to the conditions. We conclude that apolipoprotein D is only one of several plasma proteins, which contain two homologous polypeptide antigenic sites, recognized by monoclonal antibodies and also by a specific goat antiserum. Apolipoprotein D had the least Mr of these proteins.