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H S Mason

Publications and source records attributed to H S Mason.

At least 37 records · Page 2Linked to original sources
PubMed1995

Oral immunization with a recombinant bacterial antigen produced in transgenic plants.

The binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) is a highly active oral immunogen. Transgenic tobacco and potato plants were made with the use of genes encoding LT-B or an LT-B fusion protein with a microsomal retention sequence. The plants expressed the foreign peptides, both of which formed oligomers that bound the natural ligand. Mice immunized by gavage produced serum and gut mucosal anti-LT-B immunoglobulins that neutralized the enterotoxin in cell protection assays. Feeding mice fresh transgenic potato tubers also caused oral immunization.

Administration, Oral↗
PubMed1995

Immunogenicity of transgenic plant-derived hepatitis B surface antigen.

The focus of the Children's Vaccine Initiative is to encourage the discovery of technology that will make vaccines more readily available to developing countries. Our strategy has been to genetically engineer plants so that they can be used as inexpensive alternatives to fermentation systems for production of subunit antigens. In this paper we report on the immunological response elicited in vivo by using recombinant hepatitis B surface antigen (rHBsAg) purified from transgenic tobacco leaves. The anti-hepatitis B response to the tobacco-derived rHBsAg was qualitatively similar to that obtained by immunizing mice with yeast-derived rHBsAg (commercial vaccine). Additionally, T cells obtained from mice primed with the tobacco-derived rHBsAg could be stimulated in vitro by the tobacco-derived rHBsAg, yeast-derived rHBsAg, and by a synthetic peptide that represents part of the a determinant located in the S region (139-147) of HBsAg. Further support for the integrity of the T-cell epitope of the tobacco-derived rHBsAg was obtained by testing the ability of the primed T cells to proliferate in vitro after stimulation with a monoclonal anti-idiotype and an anti-idiotype-derived peptide, both of which mimic the group-specific a determinant of HBsAg. In total, we have conclusively demonstrated that both B- and T-cell epitopes of HBsAg are preserved when the antigen is expressed in a transgenic plant.

Amino Acid Sequence↗
PubMed1995

Transgenic plants as vaccine production systems.

Transgenic plants that express foreign proteins with industrial or pharmaceutical value represent an economical alternative to fermentation-based production systems. Specific vaccines have been produced in plants as a result of the transient or stable expression of foreign genes. It has recently been shown that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. Transgenic potato tubers expressing a bacterial antigen stimulated humoral and mucosal immune responses when they were provided as food. These results provide 'proof of concept' for the use of plants as a vehicle to produce vaccines.

Animals↗
PubMed1993

Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter.

Soybean vspB encodes a highly expressed vegetative storage protein-acid phosphatase. In soybean, vspB expression is stimulated by methyl jasmonate (MeJA) and sugars. The vspB promoter was studied by transforming tobacco with fusions of 5' noncoding vspB DNA and the gene encoding beta-glucuronidase (GUS). Constructs containing 833 bp of vspB 5' DNA showed high expression of GUS in stems, leaf veins and trichomes, sepals, and pollen. Sucrose (0.2 M) and MeJA (10(-5) M) increased gene expression when applied to leaf tissue. Deletion of the region -787 to -520 with respect to the transcription initiation site rendered the vspB promoter noninducible by MeJA but still sucrose responsive. This result indicates that DNA elements capable of modulating vspB by MeJA can be separated from carbon response elements. Further 5' end deletion from -520 to -403 or 3' end deletion from -165 to -289 removed DNA sequences involved in carbon modulation of gene expression. A DNA domain that mediates the MeJA response was further localized to a 50-bp region between -535 and -585. This domain when fused to a cauliflower mosaic virus (CaMV) 35S truncated (-88) promoter makes the CaMV promoter responsive to MeJA. The MeJA-responsive domain contains a G-box motif (CACGTG) and a C-rich sequence. A similar 50-bp DNA region is present in the putative promoter of vspA. Related sequences are located in a wound- and MeJA-responsive domain of the proteinase inhibitor II gene and a UV-responsive promoter domain of chs, the gene encoding chalcone synthase that is also responsive to MeJA.

Acetates↗
PubMed1992

Expression of hepatitis B surface antigen in transgenic plants.

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.

Cloning, Molecular↗
PubMed1992

The soybean vegetative storage proteins VSP alpha and VSP beta are acid phosphatases active on polyphosphates.

The soybean vegetative storage protein genes (vspA, and vspB) are regulated in a complex manner developmentally and in response to external stimuli such as wounding and water deficit. The proteins accumulate to almost one-half the amount of soluble leaf protein when soybean plants are continually depodded and have been identified as storage proteins because of their abundance and pattern of expression in plant tissues. We have shown that purified VSP homodimers (VSP alpha and VSP beta) and heterodimers (VSP alpha/beta) possess acid phosphatase activity (alpha = 0.3-0.4 units/mg; beta = 2-4 units/mg; alpha/beta = 7-10 units/mg). Specific activities were determined by monitoring o-carboxyphenyl phosphate (0.7 mM) cleavage at pH 5.5 (VSP alpha) or pH 5.0 (VSP alpha/beta and VSP beta) in 0.15 M sodium acetate buffer at 25 degrees C. These enzymes are active over a broad pH range, maintaining greater than 40% of maximal activity from pH 4.0 to 6.5 and having maximal activity at pH 5.0-5.5. They are inactivated by sodium fluoride, sodium molybdate, and heating at 70 degrees C for 10 min. These phosphatases can liberate Pi from several different substrates, including napthyl acid phosphate, carboxyphenyl phosphate, sugar-phosphates, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, ATP, ADP, PPi, and short chain polyphosphates. VSP alpha/beta cleaved phosphoenolpyruvate, ATP, ADP, PPi, and polyphosphates most efficiently. Apparent Km and Vmax values at 25 degrees C and pH 5.0 were 42 microM and 2.0 mumol/min/mg, 150 microM and 4.2 mumol/min/mg, and 420 microM and 4.1 mumol/min/mg, for tetrapolyphosphate, pyrophosphate, and phosphoenolpyruvate, respectively.

Acid Phosphatase↗
PubMed1992

Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars.

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.

Journal Article↗
PubMed1990

Water Deficit and Abscisic Acid Cause Differential Inhibition of Shoot versus Root Growth in Soybean Seedlings : Analysis of Growth, Sugar Accumulation, and Gene Expression.

Roots often continue to elongate while shoot growth is inhibited in plants subjected to low-water potentials. The cause of this differential response to water deficit was investigated. We examined hypocotyl and root growth, polysome status and mRNA populations, and abscisic acid (ABA) content in etiolated soybean (Glycine max [L.] Merr. cv Williams) seedlings whose growth was inhibited by transfer to low-water potential vermiculite or exogenous ABA. Both treatments affected growth and dry weight in a similar fashion. Maximum inhibition of hypocotyl growth occurred when internal ABA levels (modulated by ABA application) reached the endogenous level found in the elongating zone of seedlings grown in water-deficient vermiculite. Conversely, root growth was affected to only a slight extent in low-water potential seedlings and by most ABA treatments (in some, growth was promoted). In every seedling section examined, transfer of seedlings into low-water potential vermiculite caused ABA levels to increase approximately 5- to 10-fold over that found in well-watered seedlings. Changes in soluble sugar content, polysome status, and polysome mRNA translation products seen in low-water potential seedlings did not occur with ABA treatments sufficient to cause significant inhibition of hypocotyl elongation. These data suggest that both variation in endogenous ABA levels, and differing sensitivity to ABA in hypocotyls and roots can modulate root/shoot growth ratios. However, exogenous ABA did not induce changes in sugar accumulation, polysome status, and mRNA populations seen after transfer into low-water potential vermiculite.

Journal Article↗
PubMed1990

Expression of two soybean vegetative storage protein genes during development and in response to water deficit, wounding, and jasmonic acid.

The expression of vspA and vspB genes encoding soybean vegetative storage proteins was studied during seedling development and in response to water deficit, tissue wounding, and jasmonic acid treatment. vspA and vspB encode VSP-alpha and VSP-beta, 28-kilodalton and 31-kilodalton vacuole-localized polypeptides that are 80% homologous. vspA and vspB mRNAs could be distinguished on RNA blots using 3'-end probes. vspA mRNA was threefold to sevenfold more abundant than vspB mRNA in leaves, about equal expression was observed in stems, and vspB mRNA exceeded vspA in roots. Transcripts were not detected in dry seeds but appeared in intact or excised seedling axes between 12 hr and 24 hr after initiation of imbibition. Both transcripts were highly abundant in the meristematic region of seedling stems and in developing leaves but were rare in mature stems, leaves, and roots. In situ localization showed that vsp transcripts were found throughout the hypocotyl hook but were concentrated in cells associated with the epidermis and vascular bundles. Water deficit caused increased vsp mRNA levels in leaves and stems, which suggests that inhibition of growth necessitates temporary storage of amino acids. Wounding induced primarily vspB mRNA in etiolated seedlings, whereas both vspA and vspB mRNA levels increased in wounded leaves. Jasmonic acid and methyl jasmonate were potent inducers of vsp gene expression in cell cultures, developing axes, leaves, and roots. We hypothesize that jasmonic acid levels modulate vsp mRNA abundance in vivo.

Cyclopentanes↗
PubMed1988

Light-regulated translation of chloroplast proteins. I. Transcripts of psaA-psaB, psbA, and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings.

We have previously observed (Klein, R. R., and J. E. Mullet, 1986, J. Biol. Chem. 261:11138-11145) that translation of two 65-70-kD chlorophyll a-apoproteins of Photosystem I (gene products of psaA and psaB) and a 32-kD quinone-binding protein of Photosystem II (gene product of psbA) was not detected in plastids of dark-grown barley seedlings even though transcripts for these proteins were present. In the present study it was found that nearly all of the psaA-psaB transcripts in plastids of dark-grown plants were associated with membrane-bound polysomes. Membrane-associated polysomes from plastids of dark-grown plants synthesized the 65-70-kD chlorophyll a-apoproteins at low levels when added to a homologous in vitro translation extract capable of translation elongation. However, when etioplast membranes were disrupted with detergent, in vitro synthesis of the 65-70-kD chlorophyll a-apoproteins increased to levels observed with polysomes of plastids from illuminated plants. These results suggest that synthesis of the chlorophyll a-apoproteins of Photosystem I is arrested on membrane-bound polysomes at the level of polypeptide chain elongation. In addition to the selective activation of chlorophyll a-apoprotein translation, illumination also caused an increase in chloroplast polysomes (membrane-associated and stromal) and induced a recruitment of psbA and rbcL transcripts into chloroplast polysomes. These results indicate that in conjunction with the selective activation of chlorophyll a-apoprotein elongation, illumination also caused a general stimulation of chloroplast translation initiation.

Cell Fractionation↗
PubMed1988

Polysomes, Messenger RNA, and Growth in Soybean Stems during Development and Water Deficit.

The polysome status and populations of polysomal mRNA were examined in different regions of dark-grown soybean (Glycine max [L.] Merr.) stems that contained either dividing, elongating, or mature (nongrowing) cells. There was a developmental gradient of polysome content in which the dividing tissue had the highest levels and the mature tissue the lowest. A few hours after transplanting the seedlings to vermiculite having low water content (water potential Psi(w) = -0.29 megapascals), stem growth rate decreased to 30% of well-watered controls and the polysome content decreased most in the dividing and elongating tissues. After 24 to 36 hours, stem growth and polysome content recovered gradually. In vitro translation products of polysomal mRNA from dividing, elongating or mature tissue were examined on two-dimensional gels. In well-watered controls, each of the stem regions was enriched in a small subset of the polysomal mRNA population, probably because of developmentally regulated gene expression. Exposing plants to low Psi(w) for 24 hours induced a change in the relative abundance of a small number of polysomal mRNAs in the elongating and mature tissues, but not in the dividing tissue. After 24 to 72 hours at low Psi(w), the changes in polysomal mRNA population were reversed in the elongating tissue. The data indicate that changes in stem growth at low water potential are associated with changes in polysome status and polysomal mRNA in the elongating tissue.

Journal Article↗
PubMed1985

Flavins of NADPH-cytochrome P-450 reductase: evidence for structural alteration of flavins in their one-electron-reduced semiquinone states from resonance Raman spectroscopy.

The mechanism of electron transfer from NADPH to cytochrome P-450 through FAD and FMN of the reductase is largely unknown. In this paper, we report the resonance Raman spectral properties of the oxidized and the semiquinonoid states of the flavins in the holoenzyme and the FMN-depleted forms, respectively, of detergent-solubilized rabbit liver microsomal NADPH-cytochrome P-450 reductase. The resonance Raman spectra of the oxidized forms [FAD; FMN] and [FAD;-] were essentially identical, indicating similar binding interactions of these flavins with the protein. To the contrary, the spectra of the semiquinonoid FADH. and FMNH. forms revealed significant spectral differences. Both O2-unstable species, characterized as [FADH.; FMNH2] and [FADH.;-] excited at 568.2 nm, have dominant spectral peaks at approximately 1611, 1539-1543, 1377, 1305, 1263, and 1226 cm-1. However, in the O2-stable [FAD; FMNH.] species, resonance Raman bands were located at 1611, 1532, 1388, 1304, 1268, and 1227 cm-1 when excited at the same wavelength. The approximately 10-cm-1 shifts of the 1532- and 1388-cm-1 bands suggest that the environments surrounding rings II and III of the isoalloxazines change upon reduction to semiquinonoid forms. It is proposed that N1 of FADH. (as a hydrogen-bond acceptor) and N5 of FMNH. (as donor) provide the distinguishing flavin-protein interactions in the semiquinonoid states. Furthermore, the resonance Raman spectra of the semiquinonoid species appear to be missing a number of bands assigned to ring I vibrations in the spectra of the oxidized flavins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Location of heme a on subunits I and II and copper on subunit II of cytochrome c oxidase.

A systemic study has been made of copper and heme a binding to subunits of beef heart cytochrome c oxidase. Copper and heme a were readily mobilized by ionic detergents, high ionic strengths, temperatures above 0 degrees C, thiol compounds, and gel-bound peroxides and free radicals when the subunits of the oxidase were dissociated from one another during polyacrylamide gel electrophoresis. Most subunits showed some affinity for heme a and copper under these conditions. However, in the presence of specific mixtures of ionic and nonionic detergents (e.g. 0.1% sodium dodecyl sulfate, 0.025% Triton X-100) at temperatures below 0 degrees C and in buffers of low ionic strength using 10 to 12% polyacrylamide gels preelectrophoresed for 3 days with thioglycolate, about 90% of the Cu was found on subunit II (Mr = 24,100), and heme a was found in equal amounts of subunits I (Mr = 35,800) and II. The oxidized-reduced and reduced-CO absorption spectra of these subunits resembled those of cytochrome c oxidase. It appears probable that in the native enzyme, subunit I contains heme a and subunit II contains copper and heme a. A relationship of mammalian cytochrome c oxidase to the two-subunit microbial cytochrome oxidase systems appears to exist.

Animals↗
PubMed1979

Hemoprotein quantitation in isolated hepatocytes.

Methods for quantitation of catalase, cytochromes P-450 and b5 and mitochondrial cytochromes a + a3, b561 + b566, and c + c1 in isolated hepatocytes were developed in analogy to methods established for subcellular systems and were used to measure changes in specific hemoprotein concentrations due to pretreatment and to change in incubation conditions. Pretreatment of rats with phenobarbital or 3-methylcholanthrene resulted in increased concentrations of cytochromes P-450 and b5 on a cellular basis, but had no effect on the other hemoproteins. Chronic ethanol pretreatment resulted in increased cytochrome P-450 and decreased cytochromes a + a3 concentrations. Hemoprotein concentrations in hepatocytes decreased following 4-10-h incubations in rotating round-bottom flasks. Rates of decrease were dependent upon both incubation conditions and prior in vivo treatments with phenobarbital or 3-methylcholanthrene.

Animals↗