High-level expression of the HIV entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves

Key message

The potent anti-HIV microbicide griffithsin was expressed to high levels in tobacco chloroplasts, enabling efficient purification from both fresh and dried biomass, thus providing storable material for inexpensive production and scale-up on demand.

Abstract

The global HIV epidemic continues to grow, with 1.8 million new infections occurring per year. In the absence of a cure and an AIDS vaccine, there is a pressing need to prevent new infections in order to curb the disease. Topical microbicides that block viral entry into human cells can potentially prevent HIV infection. The antiviral lectin griffithsin has been identified as a highly potent inhibitor of HIV entry into human cells. Here we have explored the possibility to use transplastomic plants as an inexpensive production platform for griffithsin. We show that griffithsin accumulates in stably transformed tobacco chloroplasts to up to 5% of the total soluble protein of the plant. Griffithsin can be easily purified from leaf material and shows similarly high virus neutralization activity as griffithsin protein recombinantly expressed in bacteria. We also show that dried tobacco provides a storable source material for griffithsin purification, thus enabling quick scale-up of production on demand.

     

Principles of biological comparison

The problems of comparative analyses in biology have been discussed, showing that a single comparative method does not exist. Several principles of comparison are elucidated, which include that comparisons do not have to possess a phylogenetic basis, can be horizontal or vertical, and can be genetic or nongenetic. Biological comparisons can be grouped roughly into historical and nonhistorical ones. Historical comparisons depend on the details of evolutionary theory, and include: (a) comparisons on which phylogenies and classifications are based; and (b) comparative studies using these phylogenies and classifications. Nonhistorical comparisons require pertinent nomological relationships between two or more variables, and include: (a) comparisons between variables used to elucidate these law-like relationships; and (b) comparisons in which unknown properties of one variable are deduced from the known properties of other variables - extrapolations made in biology.     

Characterization of Sodium-Dependent [3H]GBR-12935 Binding in Brain: A Radioligand for Selective Labelling of the Dopamine Transport Complex

High-affinity and saturable binding sites for the diphenyl-substituted piperazine derivative [3H]GBR-12935 have been characterized in crude synaptosomal membranes prepared from rat brain. The specific binding of [3H]GBR-12935 is sodium-dependent and is unevenly distributed among various brain regions, with the highest concentration of binding sites being found in the corpus striatum and nucleus accumbens. Sodium-dependent [3H]GBR-12935 binding in all other brain areas was 10% or less of the binding found in the striatum. The affinity of [3H]GBR-12935 for binding sites in the striatum is increased in the presence of Na+ but other cations, including K+, Ca2+, or Mg2+, inhibit specific binding. There is an excellent correlation (r = 0.96, p less than 0.01) between the potencies of a series of drugs in inhibiting [3H]GBR-12935 binding to striatal membranes and their potencies in inhibiting [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) uptake in synaptosomes. Agonists and antagonists of other neurotransmitter receptor or drug recognition sites have little or no effect on specific [3H]GBR-12935 binding to striatal membranes. In addition, prior intracerebroventricular administration of 6-hydroxydopamine results in a decrease in the number of specific [3H]GBR-12935 binding sites in the striatum. These data indicate that [3H]GBR-12935 is a selective radioligand of the presynaptic dopamine transport complex in brain.     

Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization

The primary structure of human C1 inhibitor was determined by peptide and DNA sequencing. The single-chain polypeptide moiety of the intact inhibitor is 478 residues (52,869 Da), accounting for only 51% of the apparent molecular mass of the circulating protein (104,000 Da). The positions of six glucosamine-based and five galactosamine-based oligosaccharides were determined. Another nine threonine residues are probably also glycosylated. Most of the carbohydrate prosthetic groups (probably 17) are located at the amino-terminal end (residues 1-120) of the protein and are particularly concentrated in a region where the tetrapeptide sequence Glx-Pro-Thr-Thr, and variants thereof, is repeated 7 times. No phosphate was detected in C1 inhibitor. Two disulfide bridges connect cysteine-101 to cysteine-406 and cysteine-108 to cysteine-183. Comparison of the amino acid and cDNA sequences indicates that secretion is mediated by a 22-residue signal peptide and that further proteolytic processing does not occur. C1 inhibitor is a member of the large serine protease inhibitor (serpin) gene family. The homology concerns residues 120 through the C-terminus. The sequence was compared with those of nine other serpins, and conserved and nonconserved regions correlated with elements in the tertiary structure of alpha 1-antitrypsin. The C1 inhibitor gene maps to chromosome 11, p11.2-q13. C1 inhibitor genes of patients from four hereditary angioneurotic edema kindreds do not have obvious deletions or rearrangements in the C1 inhibitor locus. A HgiAI DNA polymorphism, identified following the observation of sequence variants, will be useful as a linkage marker in studies of mutant C1 inhibitor genes.     

Incidence of rhesus immunisation after genetic amniocentesis.

Of 655 Rh negative women without anti-D antibody in their serum at genetic amniocentesis, 361 delivered a Rh positive infant. Prophylactic treatment with anti-D immunoglobulin was not given at amniocentesis. The women were followed prospectively, being given a screening test for antibody after amniocentesis, at delivery, and six months later. Five of these 361 women yielded a positive test result due to anti-D antibody. The immunisation rate after genetic amniocentesis was no higher than the spontaneous immunisation rate during pregnancy. Four women who had two amniocenteses in the same pregnancy and 34 women who had amniocentesis in two consecutive pregnancies with Rh positive fetuses were not immunised. Among six women with anti-D antibody in their serum before amniocentesis the titre of antibody increased in three. Amniocentesis may have worsened the outcome of these pregnancies. These results suggest that the risk of immunisation in Rh negative women is small.     

Investigations on an interspecific hybrid involving three species of the genus Beta,with special reference to isozyme polymorphism

Summary A tetraploid (2n=36) interspecific hybrid was obtained involving three species belonging to three different sections of Beta. The hybrid was highly sterile and did not show apomixis. At meiosis, up to nine bivalents were observed, most probably resulting from autosyndesis of the chromosomes of Beta lomatogona. For nine isozyme systems, individual enzyme expression was investigated in the parental species and in the hybrids. No silencing of genes or genomes was observed. In the case of some polymeric enzymes interspecific heteropolymers could be detected.     

Agrobacterium — and microprojectile — mediated viral DNA delivery into barley microspore-derived cultures

Summary Anther cultures of barley (Hordeum vulgare L. var. Igri) were used as targets for Agrobacterium-mediated DNA transfer and direct DNA uptake by particle bombardment. A wheat dwarf virus construct which can replicate to a high copy number in cereal cells provided a sensitive marker for successful DNA delivery. Although DNA delivery was achieved using both procedures, particle bombardment gave more reproducible and higher levels of infection. The ability to deliver DNA into cereal cells which have a high regeneration capacity may provide a route for stable transformation.Abbreviations WDV Wheat dwarf virus - Gus -glucuronidase - ^mA N⁶-methyladenine     

Differential expression of neural cell adhesion molecule and cadherins in pancreatic islets, glucagonomas, and insulinomas.

The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagon-producing cells (alpha-cells) and then cells that express insulin (beta-cells), somatostatin (delta-cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans in a process where cell adhesion molecules are involved. In this study we have analyzed the expression of neural cell adhesion molecule (NCAM) and cadherin molecules in neonatal, young, and adult rat islet cells as well as in glucagonomas and insulinomas derived from a pluripotent rat islet cell tumor. Whereas primary islet cells at all ages express unsialylated NCAM and E-cadherin, as do insulinomas, the glucagonomas express the polysialylated NCAM, which is characteristic for developing neurons. The glucagonomas also lose E-cadherin expression and instead express a cadherin which is similar to N-cadherin in brain. Insulinoma cells express E-cadherin but differ from primary islet cells by expressing a second cadherin molecule, which is similar to N-cadherin. The expression of NCAM and cadherin isoforms in the glucagonoma suggest that this transformed alpha-cell type has converted to an immature phenotype with strong neuronal traits, reflecting the early palce of glucagon-producing cells in the islet cell lineage. In contrast, insulinoma cells are more islet-like in their phenotype and show less neuronal traits.