bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells
Authors: Radu Caius G. , Nijagal Amar , McLaughlin Jami , Wang Li , Witte Owen N.
Published: January 2005

G2A, T cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) form a group of structurally related G protein-coupled receptors (GPCRs) originally proposed to bind proinflammatory lipids. More recent studies have challenged the identification of lipid agonists for these GPCRs and have suggested that they function primarily as proton sensors. We compared the ability of these four receptors to modulate pH-dependent responses by using transiently transfected cell lines. In accordance with previously published reports, OGR1 was found to evoke strong pH-dependent responses as measured by inositol phosphate accumulation. We also confirmed the pH-dependent cAMP production by GPR4 and TDAG8. However, we found the activity of the human G2A receptor and its mouse homolog to be significantly less sensitive to pH fluctuations as measured by inositol phosphate and cAMP accumulation. Sequence homology analysis indicated that, with one exception, the histidine residues that were previously shown to be important for pH sensing by OGR1, GPR4, and TDAG8 were not conserved in the G2A receptor. We further addressed the pH-sensing properties of G2A and TDAG8 in a cellular context where these receptors are coexpressed. In thymocytes and splenocytes explanted from receptor-deficient mice, TDAG8 was found to be critical for pH-dependent cAMP production. In contrast, G2A was found to be dispensable for this process. We conclude that members of this GPCR group exhibit differential sensitivity to extracellular protons, and that expression of TDAG8 by immune cells may regulate responses in acidic microenvironments.
A computational and experimental approach to validating annotations and gene predictions in theDrosophila melanogastergenome
Authors: Yandell Mark , Bailey Adina M. , Misra Sima , Shu ShengQiang , Wiel Colin , Evans-Holm Martha , Celniker Susan E. , Rubin Gerald M.
Published: January 2005

Five years after the completion of the sequence of theDrosophila melanogastergenome, the number of protein-coding genes it contains remains a matter of debate; the number of computational gene predictions greatly exceeds the number of validated gene annotations. We have assembled a collection of >10,000 gene predictions that do not overlap existing gene annotations and have developed a process for their validation that allows us to efficiently prioritize and experimentally validate predictions from various sources by sequencing RT-PCR products to confirm gene structures. Our data provide experimental evidence for 122 protein-coding genes. Our analyses suggest that the entire collection of predictions contains only ≈700 additional protein-coding genes. Although we cannot rule out the discovery of genes with unusual features that make them refractory to existing methods, our results suggest that theD. melanogastergenome contains ≈14,000 protein-coding genes.
BDNF modulates GABA
A
receptors microtransplanted from the human epileptic brain to
Xenopus
oocytes
Authors: Palma E. , Torchia G. , Limatola C. , Trettel F. , Arcella A. , Cantore G. , Di Gennaro G. , Manfredi M. , Esposito V. , Quarato P. P. , Miledi R. , Eusebi F.
Published: January 2005

Cell membranes isolated from brain tissues, obtained surgically from six patients afflicted with drug-resistant temporal lobe epilepsy and from one nonepileptic patient afflicted with a cerebral oligodendroglioma, were injected into frog oocytes. By using this approach, the oocytes acquire human GABA
A
receptors, and we have shown previously that the “epileptic receptors” (receptors transplanted from epileptic brains) display a marked run-down during repetitive applications of GABA. It was found that exposure to the neurotrophin BDNF increased the amplitude of the “GABA currents” (currents elicited by GABA) generated by the epileptic receptors and decreased their run-down; both events being blocked by K252A, a neurotrophin tyrosine kinase receptor B inhibitor. These effects of BDNF were not mimicked by nerve growth factor. In contrast, the GABA
A
receptors transplanted from the nonepileptic human hippocampal uncus (obtained during surgical resection as part of the nontumoral tissue from the oligodendroglioma margins) or receptors expressed by injecting rat recombinant α1β2γ2 GABA
A
receptor subunit cDNAs generated GABA currents whose time-course and run-down were not altered by BDNF. Loading the oocytes with the Ca
2+
chelator 1,2-bis(2-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetate-acetoxymethyl ester (BAPTA-AM), or treating them with Rp-8-Br-cAMP, an inhibitor of the cAMP-dependent PKA, did not alter the GABA currents. However, staurosporine (a broad spectrum PK inhibitor), bisindolylmaleimide I (a PKC inhibitor), and U73122 (a phospholipase C inhibitor) blocked the BDNF-induced effects on the epileptic GABA currents. Our results indicate that BDNF potentiates the epileptic GABA
A
currents and antagonizes their use-dependent run-down, thus strengthening GABAergic inhibition, probably by means of activation of tyrosine kinase receptor B receptors and of both PLC and PKC.
Host population persistence in the face of introduced vector-borne diseases: Hawaii amakihi and avian malaria
Authors: Woodworth Bethany L. , Atkinson Carter T. , LaPointe Dennis A. , Hart Patrick J. , Spiegel Caleb S. , Tweed Erik J. , Henneman Carlene , LeBrun Jaymi , Denette Tami , DeMots Rachel , Kozar Kelly L. , Triglia Dennis , Lease Dan , Gregor Aaron , Smith Tom , Duffy David
Published: January 2005

The past quarter century has seen an unprecedented increase in the number of new and emerging infectious diseases throughout the world, with serious implications for human and wildlife populations. We examined host persistence in the face of introduced vector-borne diseases in Hawaii, where introduced avian malaria and introduced vectors have had a negative impact on most populations of Hawaiian forest birds for nearly a century. We studied birds, parasites, and vectors in nine study areas from 0 to 1,800 m on Mauna Loa Volcano, Hawaii from January to October, 2002. Contrary to predictions of prior work, we found that Hawaii amakihi (Hemignathus virens), a native species susceptible to malaria, comprised from 24.5% to 51.9% of the avian community at three low-elevation forests (55–270 m). Amakihi were more abundant at low elevations than at disease-free high elevations, and were resident and breeding there. Infection rates were 24–40% by microscopy and 55–83% by serology, with most infected individuals experiencing low-intensity, chronic infections. Mosquito trapping and diagnostics provided strong evidence for year-round local transmission. Moreover, we present evidence that Hawaii amakihi have increased in low elevation habitats on southeastern Hawaii Island over the past decade. The recent emergent phenomenon of recovering amakihi populations at low elevations, despite extremely high prevalence of avian malaria, suggests that ecological or evolutionary processes acting on hosts or parasites have allowed this species to recolonize low-elevation habitats. A better understanding of the mechanisms allowing coexistence of hosts and parasites may ultimately lead to tools for mitigating disease impacts on wildlife and human populations.
Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling
Authors: Takahashi Satoru , Ohshima Toshio , Cho Andrew , Sreenath Taduru , Iadarola Michael J. , Pant Harish C. , Kim Yong , Nairn Angus C. , Brady Roscoe O. , Greengard Paul , Kulkarni Ashok B.
Published: January 2005

Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.
The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases
Authors: Samuelson John , Banerjee Sulagna , Magnelli Paula , Cui Jike , Kelleher Daniel J. , Gilmore Reid , Robbins Phillips W.
Published: January 2005

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc
2
Man
9
Glc
3
. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from
Giardia
and
Plasmodium
). Fourth, dolichol-PP-GlcNAc
2
Man
5
(present in
Entamoeba
and
Trichomonas
) and dolichol-PP- and N-linked GlcNAc
2
(present in
Giardia
) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.
Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time
Authors: Churchman L. Stirling , Ökten Zeynep , Rock Ronald S. , Dawson John F. , Spudich James A.
Published: January 2005

Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. The registration between the two imaging channels is measured by using fiduciary markers, and the centers of the two probes are mapped onto the same space. Multiple measurements can be made before the fluorophores photobleach, allowing intramolecular and intermolecular distances to be tracked through time. This technique's lower resolution limit lies at the upper resolution limit of single molecule FRET (smFRET) microscopy. The instrumentation and fluorophores used for SHREC can also be used for smFRET, allowing the two types of measurements to be made interchangeably, covering a wide range of interfluorophore distances. A dual-labeled duplex DNA molecule (30 bp) was used as a 10-nm molecular ruler to confirm the validity of the method. We also used SHREC to study the motion of myosin V. We directly observed myosin V's alternating heads while it walked hand-over-hand along an actin filament.
A peptide inhibitor of cytochrome
c
/inositol 1,4,5-trisphosphate receptor binding blocks intrinsic and extrinsic cell death pathways
Authors: Boehning Darren , van Rossum Damian B. , Patterson Randen L. , Snyder Solomon H.
Published: January 2005

Apoptotic stimuli augment intracellular calcium concentration through inositol 1,4,5-trisphosphate receptors (IP
3
R) on endoplasmic reticulum calcium stores. We previously discovered an apoptotic cascade wherein cytochrome
c
binds to IP
3
R early in apoptosis, resulting in dysregulated calcium release. Here we show that cytochrome
c
binding to IP
3
R depends on a cluster of glutamic acid residues within the C terminus of the channel. A cell permeant peptide derived from this sequence displaces cytochrome
c
from IP
3
R and abrogates cell death induced by staurosporine treatment of HeLa cells and Fas ligand stimulation of Jurkat cells. Small-molecule inhibitors of cytochrome
c
/IP
3
R interactions may prove useful in treating disorders associated with inappropriate intrinsic and extrinsic apoptotic signaling.
Hamiltonian long–wave expansions for water waves over a rough bottom
Authors: Craig Walter , Guyenne Philippe , Nicholls David P. , Sulem Catherine
Published: January 2005

This paper is a study of the problem of nonlinear wave motion of the free surface of a body of fluid with a periodically varying bottom. The object is to describe the character of wave propagation in a long–wave asymptotic regime, extending the results of R. Rosales & G. Papanicolaou (1983 Stud. Appl. Math.
68
, 89–102) on periodic bottoms for two–dimensional flows.We take the point of view of perturbation of a Hamiltonian system dependent on a small scaling parameter, with the starting point being Zakharov's Hamiltonian (V. E. Zakharov 1968 J. Appl. Mech. Tech. Phys. 9, 1990–1994) for the Euler equations for water waves. We consider bottom topography which is periodic in horizontal variables on a short length–scale, with the amplitude of variation being of the same order as the fluid depth. The bottom may also exhibit slow variations at the same length–scale as, or longer than, the order of the wavelength of the surface waves. We do not take up the question of random bottom variations, a topic which is considered in Rosales & Papanicolaou (1983). In the two–dimensional case of waves in a channel, we give an alternate derivation of the effective Korteweg–de Vries (KdV) equation that is obtained in Rosales & Papanicolaou (1983). In addition, we obtain effective Boussinesq equations that describe the motion of bidirectional long waves, in cases in which the bottom possesses both short and long–scale variations. In certain cases we also obtain unidirectional equations that are similar to the KdV equation. In three dimensions we obtain effective three–dimensional long–wave equations in a Boussinesq scaling regime, and again in certain cases an effective Kadomtsev–Petviashvili (KP) system in the appropriate unidirectional limit.

The computations for these results are performed in the framework of an asymptotic analysis of multiple–scale operators. In the present case this involves the Dirichlet–Neumann operator for the fluid domain which takes into account the variations in bottom topography as well as the deformations of the free surface from equilibrium.
Predator–induced synchrony in population oscillations of coexisting small mammal species
Authors: Korpimäki Erkki , Norrdahl Kai , Huitu Otso , Klemola Tero
Published: January 2005

Comprehensive analyses of long–term (1977–2003) small–mammal abundance data from western Finland showed that populations ofMicrotusvoles (field volesM.agrestisand sibling volesM.rossiaemeridionalis), bank voles (Clethrionomys glareolus) and common shrews (Sorex araneus) fluctuated synchronously in 3 year population cycles. Time–series analyses indicated that interspecific synchrony is influenced strongly by density–dependent processes. Synchrony amongMicrotusand bank voles appeared additionally to be influenced by density–independent processes. To test whether interspecific synchronization through density–dependent processes is caused by predation, we experimentally reduced the densities of the main predators of small mammals in four large agricultural areas, and compared small mammal abundances in these to those in four control areas (2.5–3 km2) through a 3 year small–mammal population cycle. Predator reduction increased densities of the main prey species,Microtusvoles, in all phases of the population cycle, while bank voles, the most important alternative prey of predators, responded positively only in the low and the increase phase. Manipulation also increased the autumn densities of water voles (Arvicola terrestris) in the increase phase of the cycle. No treatment effects were detected for common shrews or mice. Our results are in accordance with the alternative prey hypothesis, by which predators successively reduce the densities of both main and alternative prey species after the peak phase of small–mammal population cycles, thus inducing a synchronous low phase.
Cellular fine structures and histochemical reactions in the tissue of a cypress twig preserved in Baltic amber
Authors: Koller Barbara , Schmitt Jürgen M. , Tischendorf Gilbert
Published: January 2005

A twig of a cypress plant preserved for
ca
. 45 Myr in Baltic amber was analysed by light and electron microscopy. Cross–sections of the whole plant showed an almost intact tissue of the entire stem and leaves, revealing, to our knowledge, the oldest and most highly preserved tissue from an amber inclusion reported so far. The preparations are based on a new technique of internal imbedding, whereby the hollow spaces within the inclusion are filled with synthetic resin which stabilizes the cellular structures during the sectioning procedure. Cytological stains applied to the sections reacted with cell walls and nuclei. A strong green auto–fluorescence of the cuticle and the resin canals in the leaves was observed. Transmission electron micrographs revealed highly preserved fine structures of cell walls, membranes and organelles. The results were compared with taxonomically related recent
Glyptostrobus
and
Juniperus
plants.
Wound-Inducible Biosynthesis of Phytoalexin Hydroxycinnamic Acid Amides of Tyramine in Tryptophan and Tyrosine Decarboxylase Transgenic Tobacco Lines
Authors: Guillet Gabriel , De Luca Vincenzo
Published: January 2005

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC ×TYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.
Functional Genomics of the Regulation of the Nitrate Assimilation Pathway in Chlamydomonas
Authors: González-Ballester David , de Montaigu Amaury , Higuera José Javier , Galván Aurora , Fernández Emilio
Published: January 2005

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype.
Tonoplast Intrinsic Proteins AtTIP2;1 and AtTIP2;3 Facilitate NH3 Transport into the Vacuole
Authors: Loqué Dominique , Ludewig Uwe , Yuan Lixing , von Wirén Nicolaus
Published: January 2005

While membrane transporters mediating ammonium uptake across the plasma membrane have been well described at the molecular level, little is known about compartmentation and cellular export of ammonium. (The term ammonium is used to denote both NH3 and NH4  + and chemical symbols are used when specificity is required.) We therefore developed a yeast (Saccharomyces cerevisiae) complementation approach and isolated two Arabidopsis (Arabidopsis thaliana) genes that conferred tolerance to the toxic ammonium analog methylammonium in yeast. Both genes, AtTIP2;1 and AtTIP2;3, encode aquaporins of the tonoplast intrinsic protein subfamily and transported methylammonium or ammonium in yeast preferentially at high medium pH. AtTIP2;1 expression in Xenopus oocytes increased 14C-methylammonium accumulation with increasing pH. AtTIP2;1- and AtTIP2;3-mediated methylammonium detoxification in yeast depended on a functional vacuole, which was in agreement with the subcellular localization of green fluorescent protein-fusion proteins on the tonoplast in planta. Transcript levels of both AtTIPs were influenced by nitrogen supply but did not follow those of the nitrogen-derepressed ammonium transporter gene AtAMT1;1. Transgenic Arabidopsis plants overexpressing AtTIP2;1 did not show altered ammonium accumulation in roots after ammonium supply, although AtTIP2;1 mRNA levels in wild-type plants were up-regulated under these conditions. This study shows that AtTIP2;1 and AtTIP2;3 can mediate the extracytosolic transport of methyl-NH2 and NH3 across the tonoplast membrane and may thus participate in vacuolar ammonium compartmentation.
Comparative Genomics of the Pennate Diatom Phaeodactylum tricornutum
Authors: Montsant Anton , Jabbari Kamel , Maheswari Uma , Bowler Chris
Published: January 2005

Diatoms are one of the most important constituents of phytoplankton communities in aquatic environments, but in spite of this, only recently have large-scale diatom-sequencing projects been undertaken. With the genome of the centric species Thalassiosira pseudonana available since mid-2004, accumulating sequence information for a pennate model species appears a natural subsequent aim. We have generated over 12,000 expressed sequence tags (ESTs) from the pennate diatom Phaeodactylum tricornutum, and upon assembly into a nonredundant set, 5,108 sequences were obtained. Significant similarity (E < 1E-04) to entries in the GenBank nonredundant protein database, the COG profile database, and the Pfam protein domains database were detected, respectively, in 45.0%, 21.5%, and 37.1% of the nonredundant collection of sequences. This information was employed to functionally annotate the P. tricornutum nonredundant set and to create an internet-accessible queryable diatom EST database. The nonredundant collection was then compared to the putative complete proteomes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the centric diatom T. pseudonana. A number of intriguing differences were identified between the pennate and the centric diatoms concerning activities of relevance for general cell metabolism, e.g. genes involved in carbon-concentrating mechanisms, cytosolic acetyl-Coenzyme A production, and fructose-1,6-bisphosphate metabolism. Finally, codon usage and utilization of C and G relative to gene expression (as measured by EST redundance) were studied, and preferences for utilization of C and CpG doublets were noted among the P. tricornutum EST coding sequences.
Microarray Analysis Confirms the Specificity of a Chlamydomonas reinhardtii Chloroplast RNA Stability Mutant
Authors: Erickson Brian , Stern David B. , Higgs David C.
Published: January 2005

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b  6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687–697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.
Beyond Complementation. Map-Based Cloning in Chlamydomonas reinhardtii
Authors: Rymarquis Linda A. , Handley Jocelyn M. , Thomas Mabel , Stern David B.
Published: January 2005

Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.
Faster Rubisco Is the Key to Superior Nitrogen-Use Efficiency in NADP-Malic Enzyme Relative to NAD-Malic Enzyme C4 Grasses
Authors: Ghannoum Oula , Evans John R. , Chow Wah Soon , Andrews T. John , Conroy Jann P. , von Caemmerer Susanne
Published: January 2005

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k  cat; 3.8 versus 5.7 s−1 at 25°C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)−1 in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k  cat.
Alterations in Tocopherol Cyclase Activity in Transgenic and Mutant Plants of Arabidopsis Affect Tocopherol Content, Tocopherol Composition, and Oxidative Stress
Authors: Kanwischer Marion , Porfirova Svetlana , Bergmüller Eveline , Dörmann Peter
Published: January 2005

Tocopherol belongs to the Vitamin E class of lipid soluble antioxidants that are essential for human nutrition. In plants, tocopherol is synthesized in plastids where it protects membranes from oxidative degradation by reactive oxygen species. Tocopherol cyclase (VTE1) catalyzes the penultimate step of tocopherol synthesis, and an Arabidopsis (Arabidopsis thaliana) mutant deficient in VTE1 (vte1) is totally devoid of tocopherol. Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from α-tocopherol to γ-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves. Tocopherol deficiency in vte1 resulted in the increase in ascorbate and glutathione, whereas accumulation of tocopherol in VTE1 overexpressing plants led to a decrease in ascorbate and glutathione. Deficiency in one antioxidant in vte1, vtc1 (ascorbate deficient), or cad2 (glutathione deficient) led to increased oxidative stress and to the concomitant increase in alternative antioxidants. Double mutants of vte1 were generated with vtc1 and cad2. Whereas growth, chlorophyll content, and photosynthetic quantum yield were very similar to wild type in vte1, vtc1, cad2, or vte1vtc1, they were reduced in vte1cad2, indicating that the simultaneous loss of tocopherol and glutathione results in moderate oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus.
Multiple Protein Regions Contribute to Differential Activities of YABBY Proteins inReproductive Development
Authors: Meister Robert J. , Oldenhof Harriette , Bowman John L. , Gasser Charles S.
Published: January 2005

Members of the YABBY family of putative transcription factors participate in abaxial-adaxial identity determination in lateral organs in Arabidopsis (Arabidopsis thaliana). Two YABBY genes specifically expressed in reproductive structures, CRABS CLAW (CRC) and INNER NO OUTER (INO), have additional activities, with CRC promoting nectary development and carpel fusion, and INO responding to spatial regulation by SUPERMAN during ovule development. All YABBY coding regions, except YABBY5, were able to restore outer integument growth in ino-1 mutants when expressed from the INO promoter (PRO  INO). However, INO was the only YABBY family member that responded correctly to SUPERMAN to maintain the wild-type gynoapical-gynobasal asymmetry of the outer integument. By contrast, INO, FILAMENTOUS FLOWER, and YABBY3 failed to complement crc-1 when expressed from PRO  CRC. Roles of individual regions of CRC and INO in these effects were assessed using chimeric proteins with PRO  INO and PRO  CRC and the relatively constitutive cauliflower mosaic virus PRO35S. Regions of CRC were found to contribute additively to CRC-specific functions in nectary and carpel formation, with a nearly direct relationship between the amount of CRC included and the degree of complementation of crc-1. When combined with INO sequences, the central and carboxyl-terminal regions of CRC were individually sufficient to overcome inhibitory effects of SUPERMAN within the outer integument. Reproductive phenotypes resulting from constitutive expression were dependent on the nature of the central region with some contributions from the amino terminus. Thus, the YABBY family members have both unique and common functional capacities, and residues involved in differential activities are distributed throughout the protein sequences.
Enhanced Photosynthetic Performance and Growth as a Consequence of Decreasing Mitochondrial Malate Dehydrogenase Activity in Transgenic Tomato Plants
Authors: Nunes-Nesi Adriano , Carrari Fernando , Lytovchenko Anna , Smith Anna M.O. , Ehlers Loureiro Marcelo , Ratcliffe R. George , Sweetlove Lee J. , Fernie Alisdair R.
Published: January 2005

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the mitochondrial malate dehydrogenase gene in the antisense orientation and exhibiting reduced activity of this isoform of malate dehydrogenase show enhanced photosynthetic activity and aerial growth under atmospheric conditions (360 ppm CO2). In comparison to wild-type plants, carbon dioxide assimilation rates and total plant dry matter were up to 11% and 19% enhanced in the transgenics, when assessed on a whole-plant basis. Accumulation of carbohydrates and redox-related compounds such as ascorbate was also markedly elevated in the transgenics. Also increased in the transgenic plants was the capacity to use l-galactono-lactone, the terminal precursor of ascorbate biosynthesis, as a respiratory substrate. Experiments in which ascorbate was fed to isolated leaf discs also resulted in increased rates of photosynthesis providing strong indication for an ascorbate-mediated link between the energy-generating processes of respiration and photosynthesis. This report thus shows that the repression of this mitochondrially localized enzyme improves both carbon assimilation and aerial growth in a crop species.
Arabidopsis Sphingosine Kinase and the Effects of Phytosphingosine-1-Phosphate on Stomatal Aperture
Authors: Coursol Sylvie , Le Stunff Hervé , Lynch Daniel V. , Gilroy Simon , Assmann Sarah M. , Spiegel Sarah
Published: January 2005

Sphingolipids are a major component of membrane lipids and their metabolite sphingosine-1-phosphate (S1P) is a potent lipid mediator in animal cells. Recently, we have shown that the enzyme responsible for S1P production, sphingosine kinase (SphK), is stimulated by the phytohormone abscisic acid in guard cells of Arabidopsis (Arabidopsis thaliana) and that S1P is effective in regulating guard cell turgor. We have now characterized SphK from Arabidopsis leaves. SphK activity was mainly associated with the membrane fraction and phosphorylated predominantly the Δ4-unsaturated long-chain sphingoid bases sphingosine (Sph) and 4,8-sphingadienine, and to a lesser extent, the saturated long-chain sphingoid bases dihydrosphingosine and phytosphingosine (Phyto-Sph). 4-Hydroxy-8-sphingenine, which is a major sphingoid base in complex glycosphingolipids from Arabidopsis leaves, was a relatively poor substrate compared with the corresponding saturated Phyto-Sph. In contrast, mammalian SphK1 efficiently phosphorylated Sph, dihydrosphingosine, and 4,8-sphingadienine, but not the 4-hydroxylated long-chain bases Phyto-Sph and 4-hydroxy-8-sphingenine. Surface dilution kinetic analysis of Arabidopsis SphK with Sph presented in mixed Triton X-100 micelles indicated that SphK associates with the micellar surface and then with the substrate presented on the surface. In addition, measurements of SphK activity under different assay conditions combined with phylogenetic analysis suggest that multiple isoforms of SphK may be expressed in Arabidopsis. Importantly, we found that phytosphingosine-1-phosphate, similar to S1P, regulates stomatal apertures and that its action is impaired in guard cells of Arabidopsis plants harboring T-DNA null mutations in the sole prototypical G-protein α-subunit gene, GPA1.
β-Maltose Is the Metabolically Active Anomer of Maltose during Transitory Starch Degradation
Authors: Weise Sean E. , Kim Kirsten S. , Stewart Robert P. , Sharkey Thomas D.
Published: January 2005

Maltose is the major form of carbon exported from the chloroplast at night as a result of transitory starch breakdown. Maltose exists as an α- or β-anomer. We developed an enzymatic technique for distinguishing between the two anomers of maltose and tested the accuracy and specificity of this technique using β-maltose liberated from maltoheptose by β-amylase. This technique was used to investigate which form of maltose is present during transitory starch degradation in bean (Phaseolus vulgaris), wild-type Arabidopsis (Arabidopsis thaliana), two starch deficient Arabidopsis lines, and one starch-excess mutant of Arabidopsis. In Phaseolus and wild-type Arabidopsis, β-maltose levels were low during the day but were much higher at night. In Arabidopsis plants unable to metabolize maltose due to a T-DNA insertion in the gene for the cytosolic amylomaltase, (Y. Lu, T.D. Sharkey [2004] Planta 218: 466–473) levels of α- and β-maltose were high during both the day and night. In starchless mutants of Arabidopsis, total maltose levels were low and almost completely in the α-form. We also found that the subcellular concentration of β-maltose at night was greater in the chloroplast than in the cytosol by 278 μ  m. We conclude that β-maltose is the metabolically active anomer of maltose and that a sufficient gradient of β-maltose exists between the chloroplast and cytosol to allow for passive transport of maltose out of chloroplasts at night.
Reductions of Rubisco Activase by Antisense RNA in the C4 Plant Flaveria bidentis Reduces Rubisco Carbamylation and Leaf Photosynthesis
Authors: von Caemmerer Susanne , Hendrickson L. , Quinn V. , Vella N. , Millgate A.G. , Furbank R.T.
Published: January 2005

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C3 species, Rubisco operates in a low CO2 environment, which is suboptimal for both catalysis and carbamylation. In C4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO2 atmosphere close to saturation. To explore the role of Rubisco activase in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T1 progeny required high CO2 (>1 kPa) for growth. They had very low CO2 assimilation rates at high light and ambient CO2, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO2. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T1 progeny of these four primary transformants showed that CO2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C4 photosynthetic pathway.
Tryptophan 64 → arginine polymorphism of β‐3‐adrenergic receptor in Chilean women with polycystic ovary syndrome
Authors: Pérez‐Bravo Francisco , Echiburú Bárbara , Maliqueo Manuel , Santos José Luis , Sir‐Petermann Teresa
Published: January 2005

Objective  To establish the frequency of the Trp64Arg polymorphism of the β3 adrenergic receptor (ADRB3) in women with polycystic ovary syndrome (PCOS) from a Chilean population, focusing particularly on the interaction with body weight. In addition, we evaluated the relationship of the Trp64Arg variant with other metabolic components of this syndrome.

Patients and Design  In a case–control design study, a total of 106 women with clinical and hormonal evidence of PCOS and 82 healthy women (HW) were evaluated.

Measurements  An oral glucose tolerance test (OGTT) was performed and serum glucose and insulin were measured before the glucose load and 30, 60, 90 and 120 min after. Lipid profile was determined in the basal sample. Insulin resistance was assessed by the homeostatic model assessment (HOMAIR) and insulin sensitivity index (ISI) composite. A polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis was performed to determine the Trp64Arg polymorphism of ADRB3.

Results  The frequency of the heterozygous condition was similar between PCOS and HW (39%vs. 35%). Only two subjects were homozygous for arginine, both belonging to the PCOS group and having a body mass index (BMI) > 30 kg/m2. In the crude analysis, hypothesis tests and odds ratios show that there is no evidence of association between the ADRB3 Trp64Arg variant and PCOS (P = 0·47). Moreover, when data were stratified by BMI categories, the statistical test for interaction between Trp64 carrier status and obesity was not significant (P = 0·29). This variant was present in 52% of the obese PCOS patients and 40% of the obese HW. In normal weight and obese PCOS carriers, the presence of the Trp64Arg variant was associated with high triglyceride (TG) levels. A major effect of the Trp64Arg variant on insulin resistance parameters could not be demonstrated.

Conclusions  The frequency of the Trp64Arg polymorphism was similar in healthy women and PCOS women, and a possible interaction between the effect of this variant and obesity in PCOS could not be demonstrated. However, our results showed an association between triglyceride levels and the presence of this genetic variant in PCOS women.
Mutation analysis of POUF‐1, PROP‐1 and HESX‐1 show low frequency of mutations in children with sporadic forms of combined pituitary hormone deficiency and septo‐optic dysplasia
Authors: Rainbow L. A. , Rees S. A. , Shaikh M. G. , Shaw N. J. , Cole T. , Barrett T. G. , Kirk J. M. W.
Published: January 2005

Objectives  Mutations in the genes encoding the transcription factors PROP1 and POUF‐1 (Pit‐1) have been reported as common causes of combined pituitary hormone deficiency (CPHD), and HESX1 mutations have been identified in children with septo‐optic dysplasia (SOD). There are few data on UK children. We have performed mutation analysis in a large cohort of affected children within the West Midlands region to assess the feasibility of a screening strategy for molecular diagnosis in CPHD and SOD.

Design and Patients  The three coding exons of PROP1, and six exons of POUF‐1 in 27 children from 26 families with CPHD, and three exons of HESX1 in 23 children from 22 families with SOD were directly sequenced from a well‐characterized regional cohort.

Results  We identified a C to T transition in exon 6 of POUF‐1, resulting in a known missense mutation (R271W) in a mother and daughter from one family with CPHD. We also found a novel homozygous T to C transition in exon 6 of POUF‐1, resulting in a missense mutation (F233L) in a twin with CPHD. This mutation was excluded in 100 ethnically matched control alleles. We did not identify any mutations in the PROP1 gene or HESX1. The median maternal age at delivery for the CPHD children was 27 years, compared to 21 years for the mothers of SOD children (P = 0·04).

Conclusions  Mutations in POUF‐1, PROP1 and HESX1 are rare causes of CPHD and SOD, respectively, in children from the West Midlands. In particular, we did not confirm the reported ‘hotspot’ in PROP1. A screening strategy that targets familial cases is highly likely to increase the mutation yield. The young maternal age at conception of children with SOD and potential teratogen exposure indicate the predominance of environmental factors in this condition compared with CPHD.
High prevalence of long‐term cardiovascular, neurological and psychosocial morbidity after treatment for craniopharyngioma
Authors: Pereira Alberto M. , Schmid Eva M. , Schutte Pieter J. , Voormolen Joan H. C. , Biermasz Nienke R. , Van Thiel Sjoerd W. , Corssmit Eleonora P. M. , Smit Jan W. A. , Roelfsema Ferdinand , Romijn Johannes A.
Published: January 2005

Introduction  The treatment of craniopharyngiomas is associated with long‐term morbidity.

Aim of the study  To assess the long‐term functional outcome and mortality rates after treatment for craniopharyngiomas, we audited our data with special focus on cardiovascular, neurological and psychosocial morbidity.

Patients and methods  Between 1965 and 2002, 54 consecutive patients underwent surgery for craniopharyngiomas at the Leiden University Medical Centre (LUMC). Fifteen patients (25%) received additional postoperative radiation therapy. The median follow‐up period was 10 years (range 1–37 years).

Results  Long‐term cure rate was 82% and long‐term recurrence rate 18%. Visual fields/visual acuity stabilized or improved in 74% of cases. The long‐term prevalence rate of hypopituitarism was 89%. In addition, long‐term cardiovascular, neurological and psychosocial morbidity rates were high: 22% (risk factors 57%), 49% and 47%, respectively. Female sex was an independent predictor of increased cardiovascular, neurological and psychosocial morbidity (odds ratio 3·78, P = 0·031). Ten patients (18%) died during an 828 person‐year follow‐up. The actuarial patient survival rates 5, 10 and 20 years after the initial operation were 95, 85 and 85%, respectively. The standardized mortality ratio (SMR) was 2·88 [95% confidence interval (CI) 1·35–4·99].

Conclusion  Craniopharyngioma is associated with excessive long‐term multisystem morbidity and mortality, especially in female patients, despite a high cure rate. These observations indicate that dedicated long‐term follow‐up of these patients is required. The purpose of the follow‐up should be: first, to look for recurrences and to ensure appropriate endocrine replacement, especially oestrogen replacement in premenopausal females; and second, to achieve intensive control of glucose, lipids, blood pressure and weight, as in any other patient with increased risk for cardiovascular disease.