A self-contained system for the field production of plant recombinant interleukin-10

The production of pharmaceutical proteins in plants is creating a broad spectrum of new high-value traits in traditional crop species. As the production of these recombinant proteins moves from bench to field scale, containment and the presence of unwanted secondary metabolites are significant practical issues. We have developed a hybrid male-sterile low-alkaloid tobacco (MSLA) production platform. Recombinant protein is produced in leaves that are harvested prior to flowering. If considered for direct in vivo mammalian use the low-alkaloid background genotype addresses concerns about nicotine, and male sterility further reduces the risk of gene leakage. We have applied this system to the production of human interleukin-10 (phIL-10), a contra-inflammatory cytokine with potential application in the treatment of inflammatory bowel disease and autoimmune diseases. Transgenic low-alkaloid tobacco lines properly assembled a biologically active phIL-10 homodimer. Hybrids made by crossing a single homozygous high-expressing phIL-10 line with a MSLA female were field tested in a high density production system and harvested after 30 days. Recombinant phIL-10 yields were found to be similar in the hybrids and the homozygous control. MSLA tobacco is a practical, self-contained system for the production of plant recombinant proteins.     

Functionally distinct pools of actin in secretory cells

Acid secretion by the gastric parietal cell is controlledthrough movement of vesicles containing the proton pump, theH⁺-K⁺-ATPase (HK). We have usedlatrunculin B (Lat B), which binds to monomeric actin, to investigateactin turnover in the stimulated parietal cell. In isolated gastricglands, relatively high concentrations of Lat B were required toinhibit acid accumulation (ED₅₀ ~70 µM). Culturedparietal cells stimulated in the presence of low Lat B (0.1-1µM) have reduced lamellipodia formation and some aberrant punctatephalloidin-stained structures, but translocation of HK and vacuolarswelling appeared unaffected. High Lat B (10-50 µM) resulted ingross changes in actin organization (punctate phalloidin-stainedstructures throughout the cell and nucleus) and reduced translocationof HK and vacuolar swelling. Resting parietal cells treated with highLat B showed minor effects on morphology and F-actin staining. Ifresting cells treated with high Lat B were washed immediately beforestimulation, they exhibited a normal stimulated morphology. These datasuggest distinct pools of parietal cell actin: a pool highlysusceptible to Lat B primarily involved in motile function of culturedcells; and a Lat B-resistant pool, most likely microvillar filaments,that is essential for secretion. Furthermore, the stimulation processappears to accentuate the effects of Lat B, most likely through Lat Bbinding to monomer actin liberated by the turnover of the motile actinfilament pool.

     

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.     

The fauna of anoplocephalid tapeworms in domestic and wild animals of Vietnam]

101 species of oribatid mites and 12 species of helminths--anoplocephalids, transmitted by these mites, were found out by Soviet-Vietnam studies in agroecosystems and tropical forests of northern and southern Vietnam. Helminths were recorded from graminivorous mammals as follows: horses, zebu, sheep, goats, buffaloes, deer, hares, elephant, 2 species of rates, 5 species of monkeys and 11 species of birds.     

Parasternal intercostal muscle remodeling in severe chronic obstructive pulmonary disease.

Studies in experimental animals indicate that chronic increases in neural drive to limb muscles elicit a fast-to-slow transformation of fiber-type proportions and myofibrillar proteins. Since neural drive to the parasternal intercostal muscles (parasternals) is chronically increased in patients with severe chronic obstructive pulmonary diseases (COPDs), we carried out the present study to test the hypothesis that the parasternals of COPD patients exhibit an increase in the proportions of both slow fibers and slow myosin heavy chains (MHCs). Accordingly, we obtained full thickness parasternal muscle biopsies from the third interspace of seven COPD patients (mean +/- SE age: 59 +/- 4 yr) and seven age-matched controls (AMCs). Fiber typing was done by immunohistochemistry, and MHC proportions were determined by SDS-PAGE followed by densitometry. COPD patients exhibited higher proportions of slow fibers than AMCs (73 +/- 4 vs. 51 +/- 3%; P < 0.01). Additionally, COPD patients exhibited higher proportions of slow MHC than AMCs (56 +/- 4 vs. 46 +/- 4%, P < 0.04). We conclude that the parasternal muscles of patients with severe COPD exhibit a fast-to-slow transformation in both fiber-type and MHC proportions. Previous workers have demonstrated that remodeling of the external intercostals, another rib cage inspiratory muscle, elicited by severe COPD is characterized by a slow-to-fast transformation in both fiber types and MHC isoform proportions. The physiological significance of this difference in remodeling between these two inspiratory rib cage muscles remains to be elucidated.     

Spectral shifts of mammalian ultraviolet-sensitive pigments (short wavelength-sensitive opsin 1) are associated with eye length and photic niche evolution

Retinal opsin photopigments initiate mammalian vision when stimulated by light. Most mammals possess a short wavelength-sensitive opsin 1 (SWS1) pigment that is primarily sensitive to either ultraviolet or violet light, leading to variation in colour perception across species. Despite knowledge of both ultraviolet- and violet-sensitive SWS1 classes in mammals for 25 years, the adaptive significance of this variation has not been subjected to hypothesis testing, resulting in minimal understanding of the basis for mammalian SWS1 spectral tuning evolution. Here, we gathered data on SWS1 for 403 mammal species, including novel SWS1 sequences for 97 species. Ancestral sequence reconstructions suggest that the most recent common ancestor of Theria possessed an ultraviolet SWS1 pigment, and that violet-sensitive pigments evolved at least 12 times in mammalian history. We also observed that ultraviolet pigments, previously considered to be a rarity, are common in mammals. We then used phylogenetic comparative methods to test the hypotheses that the evolution of violet-sensitive SWS1 is associated with increased light exposure, extended longevity and longer eye length. We discovered that diurnal mammals and species with longer eyes are more likely to have violet-sensitive pigments and less likely to possess UV-sensitive pigments. We hypothesize that (i) as mammals evolved larger body sizes, they evolved longer eyes, which limited transmittance of ultraviolet light to the retina due to an increase in Rayleigh scattering, and (ii) as mammals began to invade diurnal temporal niches, they evolved lenses with low UV transmittance to reduce chromatic aberration and/or photo-oxidative damage.