Evaluation of selectable markers for obtaining stable transformants in the gramineae

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10⁻⁵ to 2 × 10⁻⁶) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.     

Ecophysiological Significance of CO(2)-Recycling via Crassulacean Acid Metabolism in Talinum calycinum Engelm. (Portulacaceae)

High levels of variability in gas exchange characteristics and degree of CAM-cycling were found in the same and different individuals of Talinum calycinum Engelm. collected from rock outcrops in Missouri. Differences in CO₂ assimilation were mostly correlated with differences in shoot conductance to CO₂ not shoot internal CO₂ concentration. As found previously, CAM acid fluctuations were evident in well-watered plants exhibiting C₃ gas exchange patterns (CAM-cycling) and also in drought-stressed plants with stomata closed, or nearly so, day and night (CAM-idling). Drought stress also resulted in rapid stomatal closure, conserving water during droughts. Maximal CO₂ uptake rates occurred below 35°C; higher temperatures induced decreases in CO₂ assimilation and conductance while shoot internal CO₂ concentrations remained similar. Plant water-use-efficiency was severely curtailed at temperatures above 30°C. Tissue acid fluctuations were the result of changes in malic acid concentrations. Calculations of the amount of water potentially conserved by CAM-cycling yielded values of approximately 5 to 44% of daytime water loss. Thus, CAM-cycling may be an important adaptation minimizing water loss by perennial succulents growing in shallow soil on rock outcrops.     

Generation and characterization of peptide-specific antibodies that inhibit von Willebrand factor binding to glycoprotein IIb-IIIa without interacting with other adhesive molecules. Selectivity is conferred by Pro1743 and other amino acid residues adjacent to the sequence Arg1744-Gly1745-Asp1746

We have generated antibodies against a synthetic peptide corresponding to the sequence of human von Willebrand factor (vWF) between residues Glu1737-Ser1750 which includes the Arg-Gly-Asp sequence common to several adhesive molecules. Two anti-peptide antibodies, one polyclonal, and one monoclonal reacted with native vWF and inhibited its binding to platelet glycoprotein (GP) IIb-IIIa, but showed negligible cross-reactivity with fibrinogen, fibronectin, and vitronectin, three other molecules that contain the sequence Arg-Gly-Asp and bind to platelets. The structural bases for the specificity of the two antibodies were evaluated by testing the ability of peptides homologous to the parent sequence, but with single amino acid substitutions, to neutralize the binding of the two antibodies to vWF. The substitution of Pro1743, the residue immediately adjacent to the Arg-Gly-Asp sequence on the amino-terminal side, with Phe resulted in a peptide that failed to interact with either antibody. Thus, Pro1743 is important for maintaining a peptide conformation recognized by two antibodies specific for the GP IIb-IIIa-binding domain of vWF. Other residues important for optimal peptide reactivity with the polyclonal antibody were Ser1742, Arg1744, and Gly1745, whereas Gly1741, Gly1745, and Asp1746, but not Arg1744, were important for reactivity with the monoclonal antibody. The epitopes of both antibodies, therefore, included at least 2 of the residues in the sequence Arg-Gly-Asp considered the common cell-binding site of adhesive molecules that interact with GP IIb-IIIa. Nevertheless, both antibodies reacted only with vWF. These studies demonstrate that peptide-specific antibodies, unlike the promiscuous GP IIb-IIIa receptor, can recognize distinctive structural characteristics of the cell-binding domain of adhesive molecules imposed by residues adjacent to the sequence Arg-Gly-Asp.     

Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide

The influence of isoquinolinesulfonamides (H-7 and H-8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n-formyl-nel-leu-phe (FNLP), leukotriene B4 (LTB4), and phorbol-12,13-dibutyrate (PDB). The order of potency was trifluoperazine greater than or equal to fluphenazine greater than H-7 = W-7 greater than H-8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W-7 markedly stimulated 45Ca+2 efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux, while H-7 and H-8 had no effect on 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W-7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H-7 and H-8 had no significant effects on PI cycle activity. In addition, W-7 by itself was able to stimulate a weak and short-lived "burst" of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short-lived FNLP- and LTB4-stimulated macrophage activation, fluphenazine and H-7 were used to evaluate the duration of FNLP- and LTB4-stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H-7 was the most specific in its action at protein kinase C, while the phenothiazines and W-7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP- and LTB4-stimulated PI cycle activity and subsequent superoxide anion production.     

Light reduces the excitation efficiency in the nss mutant of the sheep blowfly Lucilia

The nss (no steady state) phototransduction mutant of the sheep blowfly Lucilia was studied electrophysiologically using intracellular recordings. The effects of the nss mutation on the receptor potential are manifested in the following features of the light response. (a) The responses to a flash or to dim lights are close to normal, but the receptor potential decays close to the baseline level during prolonged illumination after a critical level of light intensity is reached. (b) The decline of the response is accompanied by a large reduction in responsiveness to light that recovers within 20 s in the dark. (c) The full reduction in responsiveness to light is reached when approximately 13% of the photopigment molecules are converted from rhodopsin (R) to metarhodopsin (M). (d) A maximal net pigment conversion from R to M by blue light induces persistent inactivation in the dark, without an apparent voltage response. This inactivation could be abolished at any time by M-to-R conversion with orange light. The above features of the mutant indicate that the effect of the nss mutation on the light response of Lucilia is very similar to the effects of the transient receptor potential (trp) mutation on the photoreceptor potential of Drosophila. Noise analysis and voltage measurements indicate that the decay of the receptor potential is due to a severe reduction in the rate of occurrence of the elementary voltage responses (bumps). The bumps are only slightly modified in shape and amplitude during the decline of the response to light of medium intensity. There is also a large increase in response latency during intense background illumination. These results are consistent with the hypothesis that separate, independent mechanisms determine bump triggering and bump shape and amplitude. The nss mutation affects the triggering mechanism of the bump.     

A novel glycoprotein expressed on sheep T and B lymphocytes is involved in a T cell activation pathway

We have produced a new mouse mAb that identifies a sheep T cell activation Ag. The mAb B5-5 is specific for low m.w. components on nearly all sheep thymocytes and peripheral T and B lymphocytes but does not label immature B cells in Peyer's patches or germinal centers. After cross-linking of target structures either directly by plastic-bound mAb or indirectly using anti-Ig reagents, peripheral T cells, but not thymocytes or peripheral B cells, were activated. IL-2 was secreted by T cells after cross-linking and activation was strongly augmented in the presence of PMA. The addition of soluble B5-5 mAb to mitogen-stimulated cultures of sheep lymphocytes resulted in a suppression of PHA responses and augmentation of PWM responses and had a variable effect on Con A responses but had no effect on LPS- or protein A-induced proliferation. When added to alloantigen-stimulated cultures, B5-5 augmented the proliferative response. The B5-5 membrane component consists of 14- to 19-kDa glycoproteins but the banding patterns obtained during SDS-PAGE analysis of 125I-labeled Ag differed between thymocytes, peripheral T cells, and peripheral B cells. On the basis of its range of expression on lymphoid cells and known biochemical and functional properties, we conclude that the B5-5 component on sheep lymphocytes is different from T cell activation Ag in other species.     

Idiotype stimulation of T lymphocytes from Trypanosoma cruzi-infected patients

Anti-Trypanosoma cruzi epimastigote antibodies (anti-epi) from pooled and individual sera from patients with chronic Chagas' disease were purified on immunoaffinity columns of epimastigotes antigens (epi) coupled to activated Sepharose 4B. SDS-PAGE analysis of purified anti-epi preparations showed only the presence of human IgG H and L chains. These antibodies preparations showed similar Western blotting profiles as the sera pools from which they originated. The main polypeptides recognized by anti-epi had apparent molecular masses 31, 46, 51, 75 and 85 kDa. No difference in these patterns were detected between anti-epi from pooled sera of cardiac (anti-epiC) and indeterminate (anti-epiI) clinical forms. Anti-epi preparations (20 to 60 micrograms/ml) of pooled and individual sera stimulated proliferation of homologous and autologous PBMN or T-lymphocyte-enriched population. The stimulatory ability was dependent upon the PBMN-anti-epi combinations. There is no direct correlation between the level of PBMN response to epi and anti-epi stimuli. Comparison of the stimulatory activities of anti-epiC vs anti-epiI on PBMN of either cardiac or indeterminate group of patients indicate that anti-epiC is significantly more active than anti-epiI (p less than 0.025). These data demonstrate the presence of auto-anti-idiotypic-T cells in chagasic patients and lead to the possibility that idiotype/anti-idiotype interactions may play a role in determining the pathogenesis of chagasic cardiopathy.     

Effect of external gamma radiation on shortening the life expectancy and rates of the formation of absorbed doses from incorporated 239Pu in dogs

External gamma irradiation of dogs with doses of 103.2 and 51.6 mC/kg combined with the effect of inhaled plutonium-239 accelerates the formation of absorbed doses in secondary organs of the radionuclide deposition by 41.7 and 2.4 times, respectively, whereas the dose of 25.8 mC/kg is ineffective. As estimated by the rate of 239Pu accumulation and by the life span shortening, the minimum effective and the maximum ineffective doses are 104.8 and 80.5 cGy and 89.2 and 79.2 cGy, respectively.     

Applied anatomy of the anterolateral femoral flap

A study of the source of the blood supply to the anterolateral femoral flap was carried out on 42 lower limbs of adult cadavers (among them 35 cadavers with injection of red latex and 1 with india ink into the arteries and 6 vascular cast specimens), and the surface locations of the vascular pedicle were detected on 50 healthy adults. It was found that the descending branch of the lateral circumflex femoral vessel was an ideal axial vessel. There are constant perforating branches of the myocutaneous artery or cutaneous branches from the intermuscular space to the anterolateral femoral skin. The area extends about 12 x 30 cm. Within the flap, the anterior branch of the anterolateral cutaneous nerve of the high is located. This flap has been widely used for free transplantation in China since 1983 with satisfactory results.