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L McIntosh

Publications and source records attributed to L McIntosh.

71 records · Page 4Linked to original sources
PubMed1988

Occurrence of alternative respiratory capacity in soybean and pea.

Capacity for the alternative respiratory pathway was assessed in leaf and root tissue of male-sterile and fertile soybean (Glycine max [L.] Merr.) plants and in leaf, embryonic axis, and epicotyl tissue as well as isolated mitochondria of pea (Pisum sativum L.) by measurement of oxygen uptake in the presence and absence of KCN and salicylhydroxamic acid. Male-sterile and fertile soybean tissues showed similar responses to the inhibitors, and both possessed a capacity for alternative respiration. We also found that tissue and isolated mitochondria from ;Progress No. 9' pea possessed alternative respiratory capacity similar to that of ;Alaska' pea.

Journal Article↗
PubMed1987

Identification of the alternative terminal oxidase of higher plant mitochondria.

In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed.

Journal Article↗
PubMed1987

Construction of an Obligate Photoheterotrophic Mutant of the Cyanobacterium Synechocystis 6803 : Inactivation of the psbA Gene Family.

psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertation of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic D1 polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.

Journal Article↗
PubMed1986

Characterization and Solubilization of the Alternative Oxidase of Sauromatum guttatum Mitochondria.

The alternative oxidase activity of Sauromatum guttatum spadix mitochondria has been investigated as to its developmental expression and tissue localization. Mitochondria rich in alternative oxidase activity were found in a yellow cortex tissue present to varying degrees within the appendix, male floral, and sterile floral regions of the spadix. During a 5-day period just prior to anthesis, the alternative oxidase activity present in the appendix region was found to increase over 10-fold. On the following day when the appendix region becomes thermogenic, cytochrome oxidase activity was found to decrease by 92%, effectively forcing electron flow through the alternative oxidase. A procedure for efficient solubilization of the alternative oxidase from appendix region mitochondria was developed. The alternative oxidase thus solubilized was sensitive to heat inactivation and trypsin digestion. The activity showed inhibition characteristics expected of the alternative oxidase in that it was sensitive to salicylhydroxamic acid and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, but relatively insensitive to KCN and antimycin A. Essential sulfhydryl group(s) were indicated by reversible inhibition by p-chloromercuribenzoic acid. The solubilized alternative oxidase was most active in the detergent N,N-bis-(3-d-glucoamidopropyl)-deoxycholamide and had a pH optimum of 6.8.

Journal Article↗
PubMed1985

Site-specific mutagenesis of ribulose-1,5-bisphosphate carboxylase/oxygenase. Evidence that carbamate formation at Lys 191 is required for catalytic activity.

Site-specific mutagenesis of a cloned gene for ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was used to examine the functional significance of carbamate activation. Lysine 191, the residue involved in carbamate formation, was replaced with a glutamate in order to mimic the anionic nature of the carbamate. The resulting enzyme was capable of binding the six-carbon transition state analog carboxyarabinitol bisphosphate, but completely lacked catalytic activity. In contrast to the wild-type enzyme, carboxyarabinitol bisphosphate binding was not stabilized by divalent metal and CO2. These observations are consistent with a proposed role for the carbamate in binding the metal required for catalysis.

Amino Acid Sequence↗
PubMed1985

Nuclear mutation leads to an accelerated turnover of chloroplast-encoded 48 kd and 34.5 kd polypeptides in thylakoids lacking photosystem II.

We have studied the synthesis and accumulation of a chloroplast-encoded 48 kd chla-reaction center protein and the 34.5 kd ;atrazine binding' protein in a nuclear maize mutant which fails to assemble photosystem II reaction centers. The failure of these polypeptides to accumulation in mutant thylakoids is not due to direct nuclear control over their synthesis but is rather due to their specific, accelerated turnover from the thylakoid membrane. The accelerated turnover of these polypeptides in mutant thylakoids is largely independent of illumination conditions, as accelerated turnover occurs in the dark as well as in the light. In contrast to wild type, the 48 kd and 34.5 kd polypeptides are preferentially associated with stroma, rather than grana, lamellae in mutant membranes, suggesting that turnover occurs before these polypeptides become enriched in the grana. The nucleus thus plays a role in the stabilization of these chloroplast-encoded photosystem II reaction center polypeptides.

Journal Article↗
PubMed1985

Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia coli.

We have placed the genes encoding ribulose-bisphosphate carboxylase/oxygenase from the Anabaena 7120 operon under transcriptional control of the lac promoter carried on the Escherichia coli plasmid pUC19. The genes encoding both the large and small subunit polypeptides (rbcL and rbcS) are transcribed and translated so that approximately 0.6% of the soluble protein in E. coli extracts is a fully functional holoenzyme with a sedimentation coefficient of approximately 18S, which contains stoichiometric amounts of the two subunits. However, expression of the large subunit polypeptide vastly exceeds that of the small subunit because the majority of transcripts terminate in the intergenic region between the rbcL and rbcS genes. As a result, excess large subunit is synthesized and accumulates in E. coli as an insoluble and catalytically inactive form. Because small subunit is found only in the high molecular weight soluble form of ribulosebisphosphate carboxylase/oxygenase, we propose that the small subunit promotes assembly of the hexadecameric form of the enzyme via heterodimers of large and small subunits.

Journal Article↗
PubMed1982

The maize chloroplast genes for the beta and epsilon subunits of the photosynthetic coupling factor CF1 are fused.

We have cloned and sequenced the maize chloroplast genome fragment Eco RI e which contains the 2.2 kb transcript previously reported (Link, G. and Bogorad, L. (1980) Proc. Nat. Acad. Sci. 77 6821-6825) to lie next to the maize gene for the large subunit of ribulose bisphosphate carboxylase (LS) and to be transcribed divergently. Immunochemical and sequencing data show that the gene codes for the beta subunit of the maize chloroplast coupling factor complex (CF1). The derived amino acid sequence is highly homologous to that of the corresponding E. coli protein (Saraste et al. (1981) Nucleic Acids Res. 9 5287-5296). The last base of the codon for the terminal lysine residue of the beta subunit of CF1 is the first base of the codon for the initiating methionine of an open reading frame whose derived amino acid composition and size closely match that reported for the epsilon subunit (Binder et al. (1978) J. Biol. Chem. 253 3094-3100). The close coupling of the two genes may serve to in sure their stoichiometric production.

Base Sequence↗
PubMed1981

Differential transcription in vivo and in vitro of two adjacent maize chloroplast genes: The large subunit of ribulosebisphosphate carboxylase and the 2.2-kilobase gene.

The transcription of cloned maize plastid DNA sequences in vitro by maize plastid DNA-dependent RNA polymerase has been studied to expose the roles of the enzyme, polypeptide cofactors, and DNA sequences in the regulation of gene expression. The 4.35-kilobase pair BamHI fragment 9 carries the maize plastid gene for the large subunit of ribulosebisphosphate carboxylase and part of the gene for a 2.2-kilobase RNA. These two genes are separated by approximately 330 base pairs and are transcribed divergently. Transcripts of the gene for the large subunit of ribulosebisphosphate carboxylase are abundant in bundle sheath cells of maize leaves and we show here that transcripts of the 2.2-kilobase RNA gene are present in both mesophyll cells and the adjacent bundle sheath cells. In vitro, in the presence of the S factor, maize chloroplast DNA-dependent RNA polymerase produces a transcript of the gene for the large subunit of ribulose-bisphosphate carboxylase with a 5' terminus like that of the corresponding mRNA isolated from plastids, transcribes chloroplast DNA sequences of Bam fragment 9 in a chimeric plasmid in preference to the vehicle RSF 1030 and, in a ratio of 3:1, preferentially transcribes the gene for the large subunit of ribulosebisphosphate carboxylase over the 2.2-kilobase RNA gene from supercoiled chimeric plasmid DNA.

Journal Article↗
PubMed1981

Identification of the triazine receptor protein as a chloroplast gene product.

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed "B." The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[(14)C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [(35)S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[(14)C]atrazine, [(35)S]methionine in vivo, or [(35)S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide.

Journal Article↗
PubMed1978

Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation.

Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described. It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA. Sauromatum but not Olisthodiscus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure. Nuclease levels were monitored by coincubating [3H]-labeled Escherichia coli ribosomal RNA with the experimental RNA samples. The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.

Electrophoresis↗
PubMedDate not supplied

Analysis of HIV-1 in the cervicovaginal secretions and blood of pregnant and nonpregnant women.

OBJECTIVES: To detect HIV-1 in cellular and acellular fractions of cervicovaginal secretions obtained by cervicovaginal lavage (CVL) and evaluate viral genotypes in the HIV-1-positive CVL samples. STUDY DESIGN/METHODS: This study consists of 37 HIV-1-seropositive pregnant and nonpregnant women from the United States. A total of 63 paired CVL and blood samples were collected. HIV-1 DNA from cervical cells (CC) and virion RNA from cervical supernatant (CS) was detected by gag polymerase chain reaction (PCR) assays. The HIV-1 genotypes were determined by analyzing the nested PCR-amplified V3 region sequences of the HIV-1 gp120 envelope gene. RESULTS: Within this cohort, 95% of the women were on single or combination antiretroviral therapy. Of the pregnant women, 63% of samples had HIV-1 viral DNA in the CC, and 29% of samples were positive for viral RNA in the CS. Among nonpregnant women, 71% of samples were positive for HIV-1 DNA in CC, and 46% of samples tested positive for virion RNA in CS. Plasma viral load ranged between 10,000 and 100,000 copies/mL and showed significant correlation with the detection of HIV-1 RNA in the CVL; this relation was less apparent with viral DNA in CC. The viral blood and CVL specimens were further analyzed by evaluating the genotypes of HIV-1 variants. In most patients, a high degree of similarity was observed between the viral sequences derived from blood and CVL samples. Two patients demonstrated closely related but somewhat distinct genotypic variants in CVL and blood. One subject showed clear compartmentalization in which distinct viral genotypes were observed in CVL and blood. Based on V3 loop analyses of gp120, with one exception, the cervicovaginal secretions harbored viral populations with a macrophage (CCR5)-tropic phenotype. CONCLUSIONS: This study demonstrates the unique characteris tics of HIV-1 strains in the genital secretions of a relatively large cohort of HIV-1-infected women in the United States. These results are important for further analysis of HIV-1 transmission and pathogenesis in vivo and for rational vaccine design.

Adult↗
PubMedDate not supplied

Information, discrimination and divergence in cytology. IV. Quality control in diagnostic cytology.

The performance of diagnostic cytology on Papanicolaou smears can be periodically monitored by calculating the total discrimination or the total divergence of the cytologic diagnoses against the histologic diagnoses on samples obtained by colposcope-directed biopsies. Using these measures, the annual performances of the Gynecologic Cytology Laboratory of the University of Minnesota between 1980 and 1988 were retrospectively analyzed. For those years, the total discrimination and total divergence behaved similarly and were sensitive to the performance of the total system, including specimen sampling errors and laboratory precision. The lowest limits of the permissible range of the total discrimination and total divergence were 0.15 and -1.21 decits, respectively, for a single-slide Papanicolaou test if an 80% "hit" rate was accepted as the lowest threshold for each category. The optimal numbers of category-states were not a sensitive indicator of the quality of a laboratory; i.e., the optimal number of diagnostic categories remained at three throughout the period studied.

Female↗