Biomedical subjects

F G Plumley

Publications and source records attributed to F G Plumley.

12 recordsLinked to original sources

PubMed2001

Contribution of aerobic photoheterotrophic bacteria to the carbon cycle in the ocean.

The vertical distribution of bacteriochlorophyll a, the numbers of infrared fluorescent cells, and the variable fluorescence signal at 880 nanometers wavelength, all indicate that photosynthetically competent anoxygenic phototrophic bacteria are abundant in the upper open ocean and comprise at least 11% of the total microbial community. These organisms are facultative photoheterotrophs, metabolizing organic carbon when available, but are capable of photosynthetic light utilization when organic carbon is scarce. They are globally distributed in the euphotic zone and represent a hitherto unrecognized component of the marine microbial community that appears to be critical to the cycling of both organic and inorganic carbon in the ocean.

Aerobiosis↗

PubMed2000

Comparison of spectrofluorometric and HPLC methods for the characterization of fecal porphyrins in river otters.

A spectrofluorometric method (B. Grandchamp et al., 1980, Biochem. Biophys. Acta 629, 577-586) developed for the determination of amounts of uroporphyrin I (Uro I), coproporphyrin III (Copro III), and protoporphyrin IX (Proto IX) in skin fibroblasts was compared with a high-performance liquid chromatography (HPLC) method for the analysis of porphyrins in fecal samples of river otters (Lutra canadensis). Heptacarboxylate porphyrin I and coproporphyrin I, two porphyrins determined to be critical in defining the porphyrin profile in fecal samples of river otters with the HPLC method, contributed substantially to the calculation of the concentrations of Uro I and Copro III, respectively, in standard solutions of porphyrins with the spectrofluorometric method. Fluorescent components of the fecal matrix complicated the determination of the concentrations of Uro I, Copro III, and Proto IX with the spectrofluorometric method and resulted in erroneous values for the concentrations of these porphyrins compared with values determined with the HPLC method. These results indicate that the complexity of the sample, particularly with regard to the potential presence of interfering fluorescent compounds, as well as porphyrins additional to Uro I, Copro III, and Proto IX, should be considered prior to the application of the spectrofluorometric method. An alternative HPLC method developed for the rapid characterization of porphyrin profiles in fecal samples of river otters is described.

Alaska↗

PubMed1998

Oxygen-evolving diatom thylakoid membranes.

Two protocols were developed that yielded purified oxygen-evolving thylakoid membranes from the diatom Cylindrotheca fusiformis. One protocol employed sonication, while the second involved French press lysis of protoplasts formed by brief culture of cells in a cation-depleted medium. Regardless of the method of cell breakage, some damage to electron transport components occurred. For preservation of both light-dependent electron transport activity and in vivo fluorescence properties, 2 M sorbitol proved to be more effective than 1 M sorbitol, regardless of the method used for cell lysis. Thylakoids purified in 2 M sorbitol using the protoplast/French press method showed the best preservation of in vivo fluorescence emission signals and Photosystem II activity with ferricyanide was completely inhibited by DCMU. Thylakoids purified in 2 M sorbitol using sonication had higher rates of Photosystem II activity with ferricyanide, but this activity was less sensitive to DCMU. Whole-chain electron transport activity was low in all preparations. This is the first report of O2 evolution and of long-wavelength fluorescence in purified thylakoids of any chromophytic alga.

Diatoms↗

PubMed1995

One-step extraction and concentration of pigments and acyl lipids by sec-butanol from in vitro and in vivo samples.

Photosynthetic pigments and acyl lipids were simultaneously extracted and concentrated by sec-butanol. Pigments extracted with sec-butanol were indistinguishable from those extracted using acetone as determined by quantitative and qualitative HPLC. Use of sec-butanol has several advantages over conventional extraction solvents: (1) pigments are extracted directly from polyacrylamide gel slices without an elution step; (2) pigments in dilute, isolated pigment-protein complexes are extracted and concentrated without first concentrating the sample; (3) when necessary, the concentration factor is readily increased by addition of water; (4) sec-butanol extracts acyl lipids and vitamin K1 as effectively, but much quicker, than chloroform:methanol; (5) sec-butanol rapidly extracts and concentrates pigments from thylakoids of all plant species tested and even directly from many algal/higher plant cells, facilitating analysis of pigment biosynthetic pathways using radioactive substrates; and (6) pigments are stable in sec-butanol for several days at room temperature in the dark or for many weeks if stored at -20 degrees C in darkness. Finally, sec-butanol is preferable to ether for concentrating pigments extracted with acetone.

Butanols↗

PubMed1989

Nitrogen-dependent regulation of photosynthetic gene expression.

Nitrogen-limited Chlamydomonas reinhardtii is chlorotic and very deficient in chlorophyll a/b light-harvesting complexes (LHC). Rates of synthesis of photosynthetic proteins, but especially the LHC apoproteins, are reduced 10- to 40-fold. Moderately high levels of chloroplast transcripts accumulate in nitrogen-limited cells, and there is a correlation between chloroplast DNA levels and chloroplast mRNA abundance. In contrast, nuclear transcripts encoding LHCII and ribulose 1,5-bisphosphate carboxylase small subunits are markedly reduced. Thus, nitrogen availability affects chloroplast protein synthesis by inhibition of translation and, to a lesser extent, chloroplast DNA amplification. Regulation of nuclear-encoded photosynthetic proteins by nitrogen is achieved through mechanisms affecting transcription and/or mRNA stability.

Journal Article↗

PubMed1987

Chlorophyll antenna proteins of photosystem I: topology, synthesis, and regulation of the 20-kDa subunit of Chlamydomonas light-harvesting complex of photosystem I.

The light-harvesting complex of photosystem I (LHCI) was isolated from wild-type cells of Chlamydomonas reinhardtii; the Chl a/b-protein complex contains four major polypeptides of approximately 27, 26, 24, and 20 kDa (polypeptides 14, 15, 17.2, and 22, respectively, in the nomenclature for Chlamydomonas thylakoid proteins). Antiserum against the 20-kDa subunit of LHCI was prepared and used to determine the membrane topology, subcellular site of synthesis, and cell-cycle regulation of this polypeptide. The results indicate that the 20-kDa subunit as well as the other major LHCI polypeptides are integral membrane proteins. Moreover, protease digestion experiments reveal that the 20-kDa polypeptide is completely protected by the membrane bilayer but the 27- and 26-kDa LHCI polypeptides are exposed at the membrane surface. In vivo synthesis of the 20-kDa polypeptide is sensitive to cycloheximide but not to chloramphenicol; the form of the polypeptide recovered from in vitro translations of polyadenylated RNA is approximately 24 kDa, 4 kDa larger than the mature polypeptide. It is concluded that this LHCI polypeptide is nuclear encoded and synthesized in the cytoplasm as a higher molecular weight precursor. Synthesis of the 20-kDa polypeptide is restricted to the light period in light-dark synchronized cells. Translatable mRNA for this polypeptide accumulates during the light but levels are dramatically reduced during the dark period. Thus, synthesis of the 20-kDa subunit of LHCI appears to be transcriptionally regulated during the cell cycle.

Cell Cycle↗

PubMed1987

Reconstitution of chlorophyll a/b light-harvesting complexes: Xanthophyll-dependent assembly and energy transfer.

A method for in vitro reconstitution of the chlorophyll a/b light-harvesting complex from LiDodSO(4)/heat-denatured or acetone-extracted photosynthetic membranes has been developed. Characterization of the minimum components necessary for the functional organization of pigments in these membrane complexes reveals that xanthophylls are essential structural components.

Journal Article↗

PubMed1987

Phosphorylation of Photosystem II Components, CP43 Apoprotein, D1, D2, and 10 to 11 Kilodalton Protein in Chloroplast Thylakoids of Higher Plants.

Phosphorylated thylakoid proteins of spinach (Spinacia oleracea L.) and pea (Pisum sativum L.) were solubilized, fractionated by sucrose density gradient centrifugation, and analyzed by gel electrophoresis and crossed immunoelectrophoresis to identify the phosphoproteins. It was found that in addition to intense phosphorylation of light-harvesting chlorophyll complex II, four photosystem II components, CP43 apoprotein, D1, D2, and a 10 to 11 kilodalton protein, are substantially phosphorylated in the light. Furthermore, the CP43 apoprotein, D1 and D2 can be resolved into two electrophoretic subspecies, only one of which is phosphorylated. This indicates that only a fraction of the PSII polypeptides is phosphorylated. Finally, analysis of detergent procedures suggests that the 10 to 11 kilodalton phosphoprotein is a peripheral component of the O(2)-evolving PSII reaction center complex.

Journal Article↗

PubMed1986

Biogenesis of photosystem II complexes: transcriptional, translational, and posttranslational regulation.

The integral membrane proteins of photosystem II (PS II) reaction center complexes are encoded by chloroplast genomes. These proteins are absent from thylakoids of PS II mutants of algae and vascular plants as a result of either chloroplast or nuclear gene mutations. To resolve the molecular basis for the concurrent absence of the PS II polypeptides, protein synthesis rates and mRNA levels were measured in mutants of Chlamydomonas reinhardtii that lack PS II. The analyses show that one nuclear gene product regulates the levels of transcripts from the chloroplast gene encoding the 51-kD chlorophyll a-binding polypeptide (polypeptide 5) but is not involved in the synthesis of other chloroplast mRNAs. Another nuclear product is specifically required for translation of mRNA encoding the 32-34-kD polypeptide, D1. The absence of either D1 or polypeptide 5 does not eliminate the synthesis and thylakoid insertion of two other integral membrane proteins of PS II, the chlorophyll a-binding polypeptide of 46 kD (polypeptide 6) and the 30-kD "D1-like" protein, D2. However, these two unassembled subunits cannot be properly processed and/or are degraded in the mutants even though they reside in the membrane. In addition, pulse labeling of the nuclear mutants and a chloroplast mutant that does not synthesize D1 mRNA indicates that synthesis of polypeptide 5 and D1 is coordinated at the translational level. A model is presented to explain how absence of one of the two proteins could lead to translational arrest of the other.

Chlamydomonas↗

PubMed1986

Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae : Physical and Immunological Characterizations.

Distinctive properties are identified in the molecular structure of ribulose, 1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing algae (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrotheca fusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common.

Journal Article↗

PubMed1986

The Water Oxidation Complex of Chlamydomonas: Accumulation and Maturation of the Largest Subunit in Photosystem II Mutants.

Photosystem II particles of Chlamydomonas reinhardtii contain three extrinsic polypeptides of 29, 20, and 16 kilodaltons, whose functions are incompletely defined. We prepared a monospecific polyclonal antibody against the 29 kilodalton protein and determined that it also specifically recognizes a protein of approximately 33 kilodaltons in thylakoid membrane fractions of several vascular plants, eukaryotic algae, and a cyanobacterium. The cross-reacting 33 kilodalton protein of pea was removed from inverted thylakoid vesicles by CaCl(2) washes demonstrating the structural relationship between the Chlamydomonas polypeptide and the largest subunit of the water oxidation complex of vascular plants. Functional identity of the Chlamydomonas polypeptide was confirmed by antibody inhibition of O(2) evolution in inverted pea vesicles. In contrast to wild-type cells, only low levels of the 29 kilodalton polypeptide are recovered with purified thylakoid membranes of the mutants examined. However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex.

Journal Article↗

PubMed1983

Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate.

A method for the immunoelectrophoretic analysis of both hydrophilic and hydrophobic proteins from whole-cell extracts solubilized with 2% (w/v) sodium dodecyl sulfate (SDS) is described. For rocket immunoelectrophoresis, Triton X-100 is added to the sample before electrophoresis to sequester non-protein-bound SDS, and polyethylene glycol (PEG) is added to the antibody gel to enhance precipitin formation. With the optimal ratio of Triton X-100 to PEG, the quantitative determination of 5 ng of protein is possible. The SDS-solubilized sample can also be analyzed by crossed immunoelectrophoresis using SDS-polyacrylamide gels in the first dimension and antibody-containing agarose gels in the second. The best results are obtained when intermediate gels without nonionic detergents are used and when ionic detergents are omitted from the cathodal gel. Precipitin peaks of high quality, reproducibility, and without artifacts are obtained using antibody concentrations 5- to 50-fold lower than with other crossed-immunoelectrophoresis procedures.

Chlamydomonas↗