David B. Stern

Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats.

Dysfunctional endothelium is associated with and, likely, predates clinical complications of diabetes mellitus, by promoting increased vascular permeability and thrombogenicity. Irreversible advanced glycation end products (AGEs), resulting from nonenzymatic glycation and oxidation of proteins or lipids, are found in plasma, vessel wall, and tissues and have been linked to the development of diabetic complications. The principal means through which AGEs exert their cellular effects is via specific cellular receptors, one of which, receptor for AGE (RAGE), is expressed by endothelium. We report that blockade of RAGE inhibits AGE-induced impairment of endothelial barrier function, and reverse, in large part, the early vascular hyperpermeability observed in diabetic rats. Inhibition of AGE- and diabetes-mediated hyperpermeability by antioxidants, both in vitro and in vivo, suggested the central role of AGE-RAGE-induced oxidant stress in the development of hyperpermeability. Taken together, these data support the concept that ligation of AGEs by endothelial RAGE induces cellular dysfunction, at least in part by an oxidant-sensitive mechanism, contributing to vascular hyperpermeability in diabetes, and that RAGE is central to this pathologic process.

Hypoxia modulates the barrier and coagulant function of cultured bovine endothelium. Increased monolayer permeability and induction of procoagulant properties.

Exposure of cultured endothelium to environments with low concentrations of oxygen, in the range of those observed in pathophysiologic hypoxemic states in vivo, compromises cellular barrier and coagulant function. An atmosphere with PO2 approximately 14 mm Hg was not lethally toxic to endothelial cultures, but cells became larger and exhibited small intercellular gaps. At low oxygen concentrations, passage of macromolecular tracers through hypoxic endothelial monolayers was accelerated in a time- and dose-dependent manner, presumably by a paracellular pathway via the gaps. Cell surface coagulant properties of the endothelium were also perturbed. At PO2 approximately 14 mm Hg thrombomodulin antigen and functional activity on the cell surface were diminished by 80-90%, and Northern blots demonstrated suppression of thrombomodulin mRNA. The decrease in thrombomodulin was twice as great compared with the general decline in total protein synthesis in hypoxia. In addition, expression of a direct Factor X activator developed under hypoxic conditions; the activator was membrane-associated and expressed on the surface of intact cultures, Ca-dependent, inhibited by HgCl2 but not PMSF, and had Km approximately 25 micrograms/ml for the substrate at pH 7.4. Synthesis of the activator was blocked by inclusion of cycloheximide, but not warfarin, in the culture medium. These results demonstrate that endothelial function is perturbed in a selective manner in the presence of low concentrations of oxygen, providing insights into mechanisms which may contribute to vascular dysfunction in hypoxemic states.

Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.

Processing and degradation of chloroplast mRNA.

The conversion of genetic information stored in DNA into a protein product proceeds through the obligatory intermediate of messenger RNA. The steady-state level of an mRNA is determined by its relative synthesis and degradation rates, i.e., an interplay between transcriptional regulation and control of RNA stability. When the biological status of an organism requires that a gene products abundance varies as a function of developmental stage, environmental factors or intracellular signals, increased or decreased RNA stability can be the determining factor. RNA stability and processing have long been known as important regulatory points in chloroplast gene expression. Here we summarize current knowledge and prospects relevant to these processes, emphasizing biochemical data. The extensive literature on nuclear mutations affecting chloroplast RNA metabolism is reviewed in another article in this volume (Barkan and Goldschmidt-Clermont, this issue).

Chlamydomonas reinhardtii at the Crossroads of Genomics

Simple, experimentally tractable systems such Saccharomyces cerevisiae, Chlamydomonas reinhardtii, and Arabidopsis thaliana are powerful models for dissecting basic biological processes. The unicellular green alga C. reinhardtii is amenable to a diversity of genetic and molecular manipulations. This haploid organism grows rapidly in axenic cultures, on both solid and liquid medium, with a sexual cycle that can be precisely controlled. Vegetative diploids are readily selected through the use of complementing auxotrophic markers and are useful for analyses of deleterious recessive alleles. These genetic features have permitted the generation and characterization of a wealth of mutants with lesions in structural, metabolic and regulatory genes. Another important feature of C. reinhardtii is that it has the capacity to grow with light as a sole energy source (photoautotrophic growth) or on acetate in the dark (heterotrophically), facilitating detailed examination of genes and proteins critical for photosynthetic or respiratory function. Other important topics being studied using C. reinhardtii, many of which have direct application to elucidation of protein function in animal cells (26), include flagellum structure and assembly, cell wall biogenesis, gametogenesis, mating, phototaxis, and adaptive responses to light and nutrient environments (32, 44). Some of these studies are directly relevant to applied problems in biology, including the production of clean, solar-generated energy in the form of H2, and bioremediation of heavy metal wastes. Recent years have seen the development of a molecular toolkit for C. reinhardtii (42, 44, 66, 98, 99). Selectable markers are available for nuclear and chloroplast transformation (4, 5, 12, 13, 30, 44, 56, 82). The Arg7 (22) and Nit1 (30) genes are routinely used to rescue recessive mutant phenotypes. The bacterial ble gene (which codes for zeocin resistance [70, 112]) is an easily scored marker for nuclear transformation, and the bacterial aadA gene (which codes for spectinomycin and streptomycin resistance) is a reliable marker for chloroplast transformation (39). Nuclear transformation can be achieved by

Mitochondrial transcription initiation: promoter structures and RNA polymerases.

SummaryA diversity of promoter structuresIt is evident that tremendous diversity exists between the modes of mitochondrial transcription initiation in the different eukaryotic kingdoms, at least in terms of promoter structures. Within vertebrates, a single promoter for each strand exists, which may be unidirectional or bidirectional. In fungi and plants, multiple promoters are found, and in each case, both the extent and the primary sequences of promoters are distinct. Promoter multiplicity in fungi, plants and trypanosomes reflects the larger genome size and scattering of genes relative to animals. However, the dual roles of certain promoters in transcription and replication, at least in yeast, raises the interesting question of how the relative amounts of RNA versus DNA synthesis are regulated, possibly via cis-elements downstream from the promoters.Mitochondrial RNA polymerasesWith respect to mitochondrial RNA polymerases, characterization of human, mouse, Xenopus and yeast enzymes suggest a marked degree of conservation in their behaviur and protein composition. In general, these systems consist of a relatively non-selective core enzyme, which itself is unable to recognize promoters, and at least one dissociable specificity factor, which confers selectivity to the core subunit. In most of these systems, components of the RNA polymerase have been shown to induce a conformational change in their respective promoters and have also been assigned the role of a primase in the replication of mtDNA. While studies of the yeast RNA polymerase have suggested it has both eubacterial (mtTFB) and bacteriophage (RPO41) orgins, it is not yet clear whether these characteristics will be conserved in the mitochondrial RNA polymerases of all eukaryotes.mtTFA-mtTFB; conserved but dissimilar functionsWith respect to transcription factors, mtTFA has been found in both vertebrates and yeast, and may be a ubiquitous protein in mitochondria. However, the divergence in non-HMG portions of the proteins, combined with differences in promoter structure, has apparently relegated mtTFA to alternative, or at least non-identical, physiological roles in vertebrates and fungi. The relative ease with which mtTFA can be purified (Fisher et al. 1991) suggests that, where present, it should be facile to detect. mtTFB may represent a eubacterial sigma factor adapted for interaction with the mitochondrial RNA polymerase. In plants, sigma-like factors capable of interacting with a eubacterial polymerase are found in chloroplasts (Lerbs et al. 1988; Tiller and Link 1993; Troxler et al. 1994) raising the possibility that a gene family in plants contributes transcriptional factors to both mitochondria and chloroplasts. In coming years, we can expect a more detailed analysis of RNA polymerases, accessory factors and promoter structures, which will lead to a better understanding of the different modes of mitochondrial transcription initiation in eukaryotic species and the evolutionary relationships between them.

Functional Analysis of Two Maize cDNAs Encoding T7-like RNA Polymerases

We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of ~100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription–polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

The Chloroplast Gene ycf9 Encodes a Photosystem II (PSII) Core Subunit, PsbZ, That Participates in PSII Supramolecular Architecture

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

RNR1, a 3′–5′ exoribonuclease belonging to the RNR superfamily, catalyzes 3′ maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana

Arabidopsis thaliana chloroplasts contain at least two 3′ to 5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3′ processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5′ and 3′ ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3′ extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis

The expression of the chloroplast genome requires specialized proteins that are coded in the nucleus and imported into the organelle. We have identified such a protein that binds the leading end of the mRNA for the most abundant chloroplast enzyme. The function of this novel stabilization factor is conserved from green algae to land plants. We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ′ untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ′ >3 ′ degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.