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Purpose: Dickkopf-1 (DKK1) is a Wnt signaling modulator promoting tumor growth, metastasis, angiogenesis, and immunosuppression by regulating innate immunity. DKK1 is over-expressed in gynecologic cancers and is associated with shortened survival. DKN-01 is a humanized monoclonal antibody with DKK1 neutralizing activity that may provide clinical benefit to patients whose tumors have overexpression of DKK1 or Wnt genetic alterations., Methods: We conducted an open-label, Phase 2 basket study with 2-stage design in patients with endometrial carcinoma (EC) and platinum-resistant/refractory epithelial ovarian cancer. DKN-01 was administered either as monotherapy or in combination with weekly paclitaxel at investigator's discretion. All patients underwent NGS testing prior to enrollment; tumor tissue was also tested for DKK1 expression by RNAscope pre-treatment and after cycle 1 if available. At least 50% of patients were required to have a Wnt signaling alteration either directly or tangentially. This publication reports results from the EC population overall and by DKK1-expression., Results: DKN-01 monotherapy and in combination with paclitaxel was more effective in patients with high DKK1-expressing tumors compared to low-expressing tumors. DKN-01 monotherapy demonstrated an objective response rate [ORR] of 25.0% vs. 0%; disease control rate [DCR] of 62.5% vs. 6.7%; median progression-free survival [PFS] was 4.3 vs. 1.8 months, and overall survival [OS] was 11.0 vs. 8.2 months in DKK1-high vs DKK1-low patients. Similarly, DKN-01 in combination with paclitaxel demonstrated greater clinical activity in patients with DKK1-high tumors compared to DKK1-low tumors: DCR was 55% vs. 44%; median PFS was 5.4 vs. 1.8 months; and OS was 19.1 vs. 10.1 months. Wnt activating mutations correlated with higher DKK1 expression. DKN-01 was well tolerated as a monotherapy and in combination with paclitaxel., Conclusions: Collectively, data demonstrates promising clinical activity of a well-tolerated drug, DKN-01, in EC patients with high tumoral DKK1 expression which frequently corresponded to the presence of a Wnt activating mutation. Future development will focus on using DKN-01 in DKK1-high EC patients in combination with immunotherapy., Competing Interests: Declaration of Competing Interest Dr. Arend participates in Data Safety Monitoring/Advisory Boards (DSMB) for Astra Zeneca, Caris Life Sciences, Clovis, Merck, Seagen, Sutro, Glaxo Smith Kline, VBL Therapeutics. Dr. Matulonis reports relationships with the Med Learning Group and participates in DSMB for: Allarity, NextCure, Alkermes, Symphogen, Trillium, Agenus, Immunogen, Novartis, Boerhinger Ingelheim, Rivkin Foundation, Ovarian Cancer Research Alliance, Clearity Foundation, and Morphosys. Dr. Kagey and Dr. Sirard are employed by and own stock in Leap Therapeutics. Dr. Hamilton reports consulting or advisory for: Pfizer (Inst), Genentech/Roche (Inst), Lilly (Inst), Puma Biotechnology (Inst), Daiichi Sankyo (Inst), Mersana (Inst), Boehringer Ingelheim (Inst), AstraZeneca (Inst), Novartis (Inst), Silverback Therapeutics (Inst), Black Diamond (Inst). Dr. Sachdev participates in DSMB for Pfizer, Immunomedics, AstraZeneca, Tempus, and Ipsen; discloses stock/options in Biosplice Therapeutics; and is employed by Biosplice Therapeutics. Dr. Duska reports royalties from JB Learning, consulting fees from UpToDate, serves as an expert law review, and participates in DSMB for Regeneron and Inovio. She reports leadership in SGO, ASCO, the NCI, and the British Journal of OBGYN. Dr. ElNaggar reports employment and stock/options with Natera. Ms. Liu and Ms. Piper were employed by LEAP Therapeutics during manuscript preparation. Dr. O'Malley participates in DSMB for: AbbVie, AdaptImmune, Agenus, Arquer Diagnostics, Arcus Biosciences, AstraZeneca, Atossa Therapeutics, Boston Biomedical, Cardiff Oncology, Celcuity, Clovis Oncology, Corcept Therapeutics, Duality Biom, Eisai, Elevar, Exelixis, Genentech, Genelux, GlaxoSmithKline, GOG Foundation, Hoffman-LaRoche, ImmunoGen, Imvax, InterVenn, INXMED, IOVANCE Biotherapeutics, Janssen, Laekna, Leap Therapeutics, Luzsana Biotechnology, Merck, Merck Sharp & Dohme, Mersana, Myriad, Novartis, NovoCure, OncoC4, Onconova, Regeneron, RepImmune, R Pharm, Roche, SeaGen, Sorrento, Sutro, Tarveda, Toray, Trillium, Umoja, Verastem, VBL Therapeutics, Vincerx, Xencor, Zentalis. All other authors report no disclosures., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The coordinated redistribution of sugars from mature "source" leaves to developing "sink" leaves requires tight regulation of sugar transport between cells via plasmodesmata (PD). Although fundamental to plant physiology, the mechanisms that control PD transport and thereby support development of new leaves have remained elusive. From a forward genetic screen for altered PD transport, we discovered that the conserved eukaryotic glucose-TOR (TARGET OF RAPAMYCIN) metabolic signaling network restricts PD transport in leaves. Genetic approaches and chemical or physiological treatments to either promote or disrupt TOR activity demonstrate that glucose-activated TOR decreases PD transport in leaves. We further found that TOR is significantly more active in mature leaves photosynthesizing excess sugars than in young, growing leaves, and that this increase in TOR activity correlates with decreased rates of PD transport. We conclude that leaf cells regulate PD trafficking in response to changing carbohydrate availability monitored by the TOR pathway., Competing Interests: The authors declare no competing interest.

The biosynthesis of seed oil and starch both depend on the supply of carbon from the maternal plant. The biochemical interactions between these two pathways are not fully understood. In the Arabidopsis mutant shrunken seed 1 (sse1)/pex16, a reduced rate of fatty acid synthesis leads to starch accumulation. To further understand the metabolic impact of the decrease in oil synthesis, we compared soluble metabolites in sse1 and wild type (WT) seeds. Sugars, sugar phosphates, alcohols, pyruvate, and many other organic acids accumulated in sse1 seeds as a likely consequence of the reduced carbon demand for lipid synthesis. The enlarged pool size of hexose-P, the metabolites at the crossroad of sugar metabolism, glycolysis, and starch synthesis, was likely a direct cause of the increased flow into starch. Downstream of glycolysis, more carbon entered the TCA cycle as an alternative to the fatty acid pathway, causing the total amount of TCA cycle intermediates to rise while moving the steady state of the cycle away from fumarate. To convert the excess carbon metabolites into starch, we introduced the Escherichia coli starch synthetic enzyme ADP-glucose pyrophosphorylase (AGPase) into sse1 seeds. Expression of AGPase enhanced net starch biosynthesis in the mutant, resulting in starch levels that reached 37% of seed weight. However, further increases above this level were not achieved and most of the carbon intermediates remained high in comparison with the WT, indicating that additional mechanisms limit starch deposition in Arabidopsis seeds.

There are two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastid nonmevalonate (nonMVA) pathway, to synthesize isopentenyl diphosphate and dimethylallyl diphosphate in plants. Carotenoids and the phytyl side chain of chlorophylls are isoprenoids derived from the plastid nonMVA pathway. All enzymes involved in the nonMVA pathway have been identified in Escherichia coli. The E. coli IspF protein catalyzes a unique cyclization reaction to convert 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate into 2-C-methyl-D-erythritol 2,4-cyclodiphosphate in the nonMVA pathway. We have characterized an Arabidopsis T-DNA insertion mutant, ispF-1, which has a null mutation in the IspF gene. Homozygous ispF-1 mutants are albino lethal and the IspF transcripts are undetectable in these plants. Moreover, the ispF-1 mutant chloroplasts are filled with vesicles instead of thylakoids. Amino acid sequence alignment reveals that the IspF proteins are highly conserved between plants and bacteria. Interestingly, expression of the Arabidopsis IspF protein can rescue the lethal phenotype of an E. coli ispF mutant. These results indicate that the Arabidopsis IspF may share similar enzymatic mechanisms with the E. coli protein.

We show that MAX1, a specific repressor of vegetative axillary bud outgrowth in Arabidopsis, acts a positive regulator of the flavonoid pathway, including 11 structural genes and the transcription factor An2. Repression of bud outgrowth requires MAX1-dependent flavonoid gene expression. As the flavonoidless state leads to lateral outgrowth in Arabidopsis, our data suggest that a flavonoid-based mechanism regulates axillary bud outgrowth and that this mechanism is under the control of MAX1. Flavonoid gene expression results in the diminished expression of auxin transporters in the bud and stem, and this, in turn, decreases the rate of polar auxin transport. We speculate that MAX1 could repress axillary bud outgrowth via regulating flavonoid-dependent auxin retention in the bud and underlying stem. Because MAX1 is implicated in synthesis of the carotenoid-derived branch regulator(s) from the root, it likely links long-distance signaling with local control of bud outgrowth.

A novel seven-transmembrane receptor family, that is comprised of human adiponectin receptors (AdipoRs) and membrane progestin receptors (mPRs) that share little sequence homology with all known G protein-coupled receptors (GPCRs), has been identified recently. Although a fish mPR has been suggested to be a GPCR, human AdipoRs seem to be structurally and functionally distinct from all known GPCRs. The identification of a novel gene family, the heptahelical protein (HHP) gene family, encoding proteins in Arabidopsis predicted to have a heptahelical transmembrane topology is reported here. There are at least five HHP genes in Arabidopsis whose encoded amino acid sequences have significant similarities to human AdipoRs and mPRs. The expression and regulation of the Arabidopsis HHP gene family has been studied here. The expression of the HHP gene family is differentially regulated by plant hormones. Steady-state levels of HHP1 mRNA are increased by treatments with abscisic acid and gibberellic acid, whereas levels of HHP2 mRNA are increased by abscisic acid and benzyladenine treatments. In addition, the expression of the HHP gene family is up-regulated by the presence of sucrose in the medium. Temperature and salt stress treatments also differentially affect the expression of the HHP genes. These novel seven-transmembrane proteins previously described in yeast and animals, and now identified in plants, may represent a new class of receptors that are highly conserved across kingdoms.

Plant isoprenoids are synthesized via two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastid nonmevalonate pathway. The Escherichia coli IspH (LytB) protein is involved in the last step of the nonmevalonate pathway. We have isolated an Arabidopsis (Arabidopsis thaliana) ispH null mutant that has an albino phenotype and have generated Arabidopsis transgenic lines showing various albino patterns caused by IspH transgene-induced gene silencing. The initiation of albino phenotypes rendered by IspH gene silencing can arise independently from multiple sites of the same plant. After a spontaneous initiation, the albino phenotype is systemically spread toward younger tissues along the source-to-sink flow relative to the initiation site. The development of chloroplasts is severely impaired in the IspH-deficient albino tissues. Instead of thylakoids, mutant chloroplasts are filled with vesicles. Immunoblot analysis reveals that Arabidopsis IspH is a chloroplast stromal protein. Expression of Arabidopsis IspH complements the lethal phenotype of an E. coli ispH mutant. In 2-week-old Arabidopsis seedlings, the expression of 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), IspD, IspE, IspF, and IspG genes is induced by light, whereas the expression of the IspH gene is constitutive. The addition of 3% sucrose in the media slightly increased levels of DXS, DXR, IspD, IspE, and IspF mRNA in the dark. In a 16-h-light/8-h-dark photoperiod, the accumulation of the IspH transcript oscillates with the highest levels detected in the early light period (2-6 h) and the late dark period (4-6 h). The expression patterns of DXS and IspG are similar to that of IspH, indicating that these genes are coordinately regulated in Arabidopsis when grown in a 16-h-light/8-h-dark photoperiod.

One of the important roles of microRNA (miRNA) is to direct the cleavage of messenger RNA (mRNA). However, the mechanisms of decay of the cleaved mRNA products is not well understood. We show that miRNA-directed cleavage products in organisms as diverse as Arabidopsis, mouse, and Epstein-Barr virus have at their 3' ends a stretch (1 to 24 nucleotides) of oligouridine posttranscriptionally added downstream of the cleavage site. This 3' uridine addition, as shown for Arabidopsis, is correlated with decapping and 5' shortening of the cleaved products, suggesting a mechanistic step in the miRNA-directed mRNA decay mechanism.

The Arabidopsis Shrunken Seed 1 (SSE1) gene encodes a homolog of the peroxisome biogenesis factor Pex16p, and a loss-of-function mutation in this gene alters seed storage composition. Two lines of evidence support a function for SSE1 in peroxisome biogenesis: the peroxisomal localization of a green fluorescent protein-SSE1 fusion protein and the lack of normal peroxisomes in sse1 mutant embryos. The green fluorescent protein-SSE1 colocalizes with the red fluorescent protein (RFP)-labeled peroxisomal markers RFP-peroxisome targeting signal 1 and peroxisome targeting signal 2-RFP in transgenic Arabidopsis. Each peroxisomal marker exhibits a normal punctate peroxisomal distribution in the wild type but not the sse1 mutant embryos. Further studies reported here were designed toward understanding carbon metabolism in the sse1 mutant. A time course study of dissected embryos revealed a dramatic rate decrease in oil accumulation and an increase in starch accumulation. Introduction of starch synthesis mutations into the sse1 background did not restore oil biosynthesis. This finding demonstrated that reduction in oil content in sse1 is not caused by increased carbon flow to starch. To identify the blocked steps in the sse1 oil deposition pathway, developing sse1 seeds were supplied radiolabeled oil synthesis precursors. The ability of sse1 to incorporate oleic acid, but not pyruvate or acetate, into triacylglycerol indicated a defect in the fatty acid biosynthetic pathway in this mutant. Taken together, the results point to a possible role for peroxisomes in the net synthesis of fatty acids in addition to their established function in lipid catabolism. Other possible interpretations of the results are discussed.

The ubiquitous bacterium Pseudomonas aeruginosa is the quintessential opportunistic pathogen. Certain isolates infect a broad range of host organisms, from plants to humans. The pathogenic promiscuity of particular variants may reflect an increased virulence gene repertoire beyond the core P. aeruginosa genome. We have identified and characterized two P. aeruginosa pathogenicity islands (PAPI-1 and PAPI-2) in the genome of PA14, a highly virulent clinical isolate. The 108-kb PAPI-1 and 11-kb PAPI-2, which are absent from the less virulent reference strain PAO1, exhibit highly modular structures, revealing their complex derivations from a wide array of bacterial species and mobile elements. Most of the genes within these islands that are homologous to known genes occur in other human and plant bacterial pathogens. For example, PAPI-1 carries a complete gene cluster predicted to encode a type IV group B pilus, a well known adhesin absent from strain PAO1. However, >80% of the PAPI-1 DNA sequence is unique, and 75 of its 115 predicted ORF products are unrelated to any known proteins or functional domains. Significantly, many PAPI-1 ORFs also occur in several P. aeruginosa cystic fibrosis isolates. Twenty-three PAPI ORFs were mutated, and 19 were found to be necessary for full plant or animal virulence, with 11 required for both. The large set of "extra" virulence functions encoded by both PAPIs may contribute to the increased promiscuity of highly virulent P. aeruginosa strains, by directing additional pathogenic functions.

GH stimulates the phosphorylation of tyrosine residues in the GH receptor (GHR), Janus kinase 2 (JAK2), and other signaling proteins in a transient manner that subsides within 1 h. To assess the possible roles of cytokine-induced Src homology domain 2 (SH2) (CIS/SOCS) proteins in these transient responses, we studied the expression and disposition of CIS/SOCS proteins in rat adipocytes, a physiological target of GH action. A tyrosine-phosphorylated protein that appears to be the GHR was coprecipitated from extracts of GH-treated adipocytes with alpha-CIS. In contrast, no tyrosine-phosphorylated adipocyte proteins were recovered after immunoprecipitation with alpha-SOCS3, although coprecipitation of GHR with SOCS3 was readily detected in extracts of 3T3-F442A fibroblasts. Interaction of GHR with CIS peaked between 2 and 10 min after adipocytes were treated with GH, when tyrosine phosphorylation of the GHR was maximal. By 60 min after GH, tyrosine phosphorylation of the GHR declined to very low levels, and its interaction with CIS was reduced correspondingly. Proteasome inhibitors prevented the decline in tyrosine-phosphorylated GHR and prolonged interaction of GHR and CIS for at least 1 h. These findings demonstrate the interaction of CIS with the GHR in vivo and suggest that CIS may enhance degradation of the receptor by a proteasomal pathway.

In bacteria, the regulatory ACT domains serve as amino acid-binding sites in some feedback-regulated amino acid metabolic enzymes. We have identified a novel type of ACT domain-containing protein family in Arabidopsis whose members contain ACT domain repeats (the "ACR" protein family). There are at least eight ACR genes located on each of the five chromosomes in the Arabidopsis genome. Gene structure comparisons indicate that the ACR gene family may have arisen by gene duplications. Northern-blot analysis indicates that each member of the ACR gene family has a distinct expression pattern in various organs from 6-week-old Arabidopsis. Moreover, analyses of an ACR3 promoter-beta-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity formed a gradient in the developing leaves and sepals, whereas low or no GUS activity was detected in the basal regions. In 2-week-old Arabidopsis seedlings grown in tissue culture, the expression of the ACR gene family is differentially regulated by plant hormones, salt stress, cold stress, and light/dark treatment. The steady-state levels of ACR8 mRNA are dramatically increased by treatment with abscisic acid or salt. Levels of ACR3 and ACR4 mRNA are increased by treatment with benzyladenine. The amino acid sequences of Arabidopsis ACR proteins are most similar in the ACT domains to the bacterial sensor protein GlnD. The ACR proteins may function as novel regulatory or sensor proteins in plants.

The role of GH in the developing fetus is poorly understood. Several studies have demonstrated a limited role for GH in late fetal life. In fact, few data are available regarding GH signal transduction in the late gestation fetus. We therefore focused on a comparison of hepatic GH signaling in near-term fetal rats [embryonic day 19 (E19)] and adult rats using a combination of in vitro studies employing hepatocytes in primary culture and in vivo studies. We found that GH receptor (GHr) binding was comparable in fetal liver and adult liver. The long isoform of the GHr underwent tyrosine phosphorylation in response to GH stimulation of E19 fetal hepatocytes in a manner similar to that seen in cultured adult hepatocytes. Furthermore, downstream signaling via the Janus kinase-2 tyrosine kinase, STAT1 (signal transducer and activator of transcription), and STAT5 was also intact in both, as demonstrated by the tyrosine phosphorylation of these signaling proteins. To confirm the relevance of these findings to the in vivo situation, GH was directly administered by ip injection to E 19 fetal and adult rats. In both cases, tyrosine phosphorylation of STAT5 was markedly and rapidly induced. Finally, transfection of E19 fetal hepatocytes with GH-responsive reporter elements [Spi2.1(-275/+85)-CAT and 8xGHRE-TKCAT] demonstrated intact transcriptional regulation. Our data indicate that GHr abundance and activity as well as downstream GH signaling are similar in the late gestation fetal rat and in the adult and that these mechanisms appear capable of supporting physiological GH functions in the developing liver.

Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative physical mapping between Arabidopsis thaliana and Brassica rapa. A set of six bacterial artificial chromosomes (BACs) representing a 431-kb contiguous region of chromosome 2 of A. thaliana was mapped on both chromosomes and DNA fibers of B. rapa. This DNA fragment has a single location in the A. thaliana genome, but hybridized to four to six B. rapa chromosomes, indicating multiple duplications in the B. rapa genome. The sizes of the fiber-FISH signals from the same BACs were not longer in B. rapa than those in A. thaliana, suggesting that this genomic region is duplicated but not expanded in the B. rapa genome. The comparative fiber-FISH mapping results support that chromosomal duplications, rather than regional expansion due to accumulation of repetitive sequences in the intergenic regions, played the major role in the evolution of the B. rapa genome.

The serine-arginine (SR)-rich splicing factors play essential roles in general splicing and regulate alternative splice site utilization in a concentration-dependent manner. SR1 is a plant homologue of the human general/alternative splicing factor SF2/ASF. We report here that alternative splicing regulates SR1 itself. Of the five detected SR1 transcripts only one encodes the full-length protein, while the other four are different variants of the essential arginine-serine-rich domain. The data suggest that SR1 pre-mRNA could be committed to two alternate splicing pathways. One, dependent on the alternative utilization of competing 3' splice sites in intron 9, generates SR1, SR1B and SR1C. The other, regulated by suppression of intron 9 5' splice site utilization, generates SR1D and SR1E. The splicing pattern and molecular structure of SR1D indicates an evolutionary conservation of splicing-based regulation between plants and vertebrates and suggests that the various isoforms perform important functions. Results from transient gene expression assays indicate that alternative splicing is not an autoregulatory mechanism used to control the transcript level of the full-length protein. The ratio of SR1/SR1B transcripts, which are generated by alternative 3' splice site utilization in intron 9, is under temperature control. The temperature-dependent increase in SR1B/SR1 ratio suggests a role of SR1B in the adaptation to high-temperature environments. In addition, based on the regulated co-expression of SR1 transcripts, it is possible that some SR1 functions could be determined by the combinatorial action of the various isoforms.

GH promptly increases cytosolic free calcium ([Ca2+]i) in freshly isolated rat adipocytes. Adipocytes deprived of GH for 3 h or longer are incapable of increasing [Ca2+]i in response to GH, but instead respond in an insulin-like manner. Insulin blocks the GH-induced increase in [Ca2+]i in GH-replete cells and stimulates the sodium pump (i.e. Na+/K+-ATPase), thereby hyperpolarizing the cell membrane. Blockade of the Na+/K+-ATPase with 100 microM ouabain reversed these effects of insulin and enabled GH to increase [Ca2+]i in GH-deprived adipocytes. Both insulin and GH activated the sodium pump in GH-deprived adipocytes, as indicated by increased uptake of 86Rb+. Decreasing availability of intracellular Na+ by blockade of Na+/K+/ 2Cl- symporters or Na+/H+ antiporters abolished the effects of both hormones on 86Rb+ uptake and enabled both GH and insulin to increase [Ca2+]i in GH-deprived adipocytes. The data suggest that hormonal stimulation of Na+/K+-ATPase activity interferes with activation of voltage-sensitive calcium channels by either membrane hyperpolarization or some unknown interaction between the sodium pump and calcium channels.

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

Mature Arabidopsis seeds are enriched in storage proteins and lipids, but lack starch. In the shrunken seed 1 (sse1) mutant, however, starch is favored over proteins and lipids as the major storage compound. SSE1 has 26 percent identity with Pex16p in Yarrowia lipolytica and complements pex16 mutants defective in the formation of peroxisomes and the transportation of plasma membrane- and cell wall-associated proteins. In Arabidopsis maturing seeds, SSE1 is required for protein and oil body biogenesis, both of which are endoplasmic reticulum-dependent. Starch accumulation in sse1 suggests that starch formation is a default storage deposition pathway.

Arabidopsis thaliana BAC T07M07 encoding the abscisic acid-insensitive 4 (ABI4) locus has been sequenced completely. It contains a 95,713-bp insert and 24 predicted genes. Most putative genes were confirmed by gel-based RNA profiling and a cluster of ABA-regulated genes was identified. One of the 24 genes, designated PP2C5, encodes a putative protein phosphatase 2C. The encoded protein was expressed in Escherichia coli, and its enzyme activity in vitro was confirmed.

We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus. The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity in vivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.

GH, in the presence of glucocorticoid, produces a delayed increase in lipolysis in rat adipose tissue, but the biochemical mechanisms that account for this action have not been established. Other lipolytic agents rapidly activate adenylyl cyclase (AC) and the resulting production of cAMP initiates a chain of reactions that culminates in the activation of hormone-sensitive lipase. We compared responses of segments of rat epididymal fat or isolated adipocytes to 30 ng/ml GH and 0.1 microg/ml dexamethasone (Dex) with 0.1 ng/ml isoproterenol (ISO), which evoked a similar increase in lipolysis. All measurements were made during the fourth hour after the addition of GH+Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant increases in cAMP were discernible in homogenates of GH+Dex-treated tissues, Rp-cAMPS (Rp-adenosine 3'5'-phosphothioate), a competitive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH+Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32P from [gamma-32P]ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH+Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH+Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K x g or a less dense membrane pellet sedimented at 100K x g, but doubled the AC activity in the 16K pellet when assayed in the absence of forskolin. To evaluate possible effects on G proteins, pellets obtained from centrifugation of adipocyte homogenates at 16K x g and 100K x g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Gi alpha2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gs alpha in the 16K pellet was unaffected by GH+Dex and was decreased (P < 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH+Dex-dependent shift of Gi alpha2 to less dense fractions as determined by both Western analysis and [32P]NAD ribosylation catalyzed by pertussis toxin. No such changes were seen in the distribution of Gs alpha or 5'-nucleotidase. Colchicine (100 microM) blocked the GH+Dex-dependent shift of Gi alpha2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH+Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Gi alpha2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.

Ribozymes are potential tools for genetic manipulation, and various naturally occurring catalytic RNAs have been dissected and used as the basis for the design of new endoribonuclease activities. While such cleaving ribozymes may work well in vitro, they have not proved to be routinely effective in depleting living cells of the chosen target RNA. Recently, trans-splicing ribozymes have been employed to repair mutant mRNAs in vivo. We have designed modified trans-splicing ribozymes with improved biological activity. These allow accurate splicing of a new 3' exon sequence into a chosen site within a target RNA, and in frame fusion of the exon can result in expression of a new gene product. These trans-splicing ribozymes contain catalytic sequences derived from a self-splicing group I intron, which have been adapted to a chosen target mRNA by fusion of a region of extended complementarity to the target RNA and precise alteration of the guide sequences required for substrate recognition. Both modifications are required for improved biological activity of the ribozymes. Whereas cleaving ribozymes must efficiently deplete a chosen mRNA species to be effective in vivo, even inefficient trans-splicing can allow the useful expression of a new gene activity, dependent on the presence of a chosen RNA. We have targeted trans-splicing ribozymes against mRNAs of chloramphenicol acetyltransferase, human immunodeficiency virus, and cucumber mosaic virus, and demonstrated trans-splicing and delivery of a marker gene in Escherichia coli cells. The improved trans-splicing ribozymes may be tailored for virtually any target RNA, and provide a new tool for triggering gene expression in specific cell types., (Copyright 1999 Academic Press.)

The 14-3-3 proteins are a group of highly conserved and widely distributed eukaryotic proteins with diverse functions. One 14-3-3 protein, AFT1 from Arabidopsis thaliana, was found to be able to activate transcription in yeast. When fused to the DNA-binding domain of a bacterial protein LexA, AFT1 can activate transcription of reporter genes that contain LexA operator sequences in their promoters. Although the in vivo function of AFT1 is not completely known, its similarity to previously identified proteins found in transcription complexes of Arabidopsis and maize suggests that AFT1 and some other 14-3-3 proteins may activate gene expression in other systems as well.

In vivo, GH and insulin usually produce opposing effects on carbohydrate and lipid metabolism in adipocytes, even though their signal transduction pathways overlap. However, when added to rat adipocytes that have been made GH deficient, GH briefly produces responses that are qualitatively like those of insulin. Subsequently, GH induces refractoriness to this acute insulin-like response, in a sense restricting its effects to a unique subset of possible physiological actions. Okadaic acid is an inhibitor of type I and IIa phosphoprotein phosphatases and affects glucose metabolism in fat cells in a manner that is reminiscent of GH. Okadaic acid initially mimics the actions of insulin, and subsequently, even after it has been removed by thorough washing, blunts the ability of adipocytes to accelerate glucose metabolism in response to insulin or GH. Because refractoriness to the insulin-like effect of GH is associated with GH-induced increases in intracellular free calcium concentrations ([Ca2+]i), we examined the effects of insulin on [Ca2+]i in okadaic acid-treated adipocytes. Adipocytes were incubated with 0.25 microM okadaic acid for 1 h, washed, and reincubated without okadaic acid for 2 h before measurement of [Ca2+]i using fura-2 as a calcium indicator. Neither GH (500 ng/ml) nor insulin (100 microU/ml) affected [Ca2+]i in cells in which glucose metabolism was stimulated, but both hormones rapidly increased [Ca2+]i in adipocytes that were refractory to insulin-like stimulation. The characteristics of the increase in [Ca2+]i produced by insulin were identical to those previously reported for GH. The effect of insulin was mimicked by the dihydropyridine calcium channel activator BayK 5552 or depolarization of the cell membrane with 30 mM KCl and was blocked by the dihydropyridine calcium channel blocker, nimodipine (100 nM), implicating activation of voltage-sensitive L-type Ca2+ channels. The increase in [Ca2+]i was also mimicked by sn-1,2-dioctanoylglycerol and blocked by inhibitors of protein kinase C (staurosporine, chelerythrine chloride, and calphostin), and D609, an inhibitor of phospholipase C, as reported for GH. Acquisition of the ability to increase [Ca2+]i in response to insulin required a lag period of at least 2 h after removal of okadaic acid and was prevented by inhibitors of RNA and protein synthesis. Adipocytes that were incubated with inhibitors of protein kinase A (KT-5720), or protein kinase C (staurosporine) along with okadaic acid also failed to increase [Ca2+]i in response to insulin. Conversely, adipocytes that were incubated with dibutyryl cAMP, methylisobutyl xanthine, or phorbol ester instead of okadaic acid increased [Ca2+]i when treated with insulin 2 h later. These results suggest that phosphorylated substrates of protein kinases A and C may mediate the transcriptional event(s) that enable adipocytes to activate L-type Ca2+ channels in response to insulin. Blockade of protein kinases A or C or removal of calcium from the incubation medium did not restore the ability of okadaic acid-treated adipocytes to increase glucose metabolism in response to insulin, nor did pretreatment of adipocytes with dibutyryl cAMP or phorbol ester decrease insulin-induced stimulation of glucose metabolism. The failure of insulin to increase glucose metabolism in okadaic acid-treated adipocytes thus cannot be ascribed to the increase in [Ca2+]i. These findings indicate that just as GH can produce an insulin-like response, so too can insulin produce a GH-like response, and highlight the need to understand how specificity of hormone action is achieved in cells that respond to different hormones that share elements of their transduction pathways.

Arabidopsis thaliana has become a model plant species for genetic studies because of its small genome and short juvenility period. However, the small chromosomes of this species are not suitable for classical cytogenetic studies. Here we demonstrate that the fluorescence in situ hybridization (FISH) technique using extended DNA fibers can be a powerful tool in the physical mapping of the A. thaliana genome. Using a refined fiber-FISH technique we were able to measure DNA clusters as long as 1.71 Mb, more than 1% of the A. thaliana genome. Several small DNA loci, including the telomeres and a dispersed repetititve DNA sequence, mi167, were also analyzed with this technique. The results show that without known adjacent DNA markers such small DNA loci cannot be mapped precisely using fiber-FISH. One of the most difficult obstacles in physical mapping by contig assembly is closing the gaps that are present between adjacent contigs. Currently available molecular techniques are not sufficient to accurately estimate the physical sizes of these gaps. We isolated bacterial artificial chromosome (BAC) clones bordering gaps 2 and 3 on the physical contig map of A. thaliana chromosome II. The BAC clones were used in fiber-FISH analysis and the physical sizes of the two gaps were estimated as 31 kb and more than 500 kb, respectively. Thus, we have demonstrated that fiber-FISH is an efficient technique for determining the physical size of gaps on molecular contig maps.

Earlier studies demonstrated that deprivation of growth hormone (GH) for >/=3 h decreased basal and maximally stimulated cytosolic Ca2+ in rat adipocytes and suggested that membrane Ca2+ channels might be decreased. Measurement of L-type Ca2+ channels in purified plasma membranes by immunoassay or dihydropyridine binding indicated a two- to fourfold decrease after 3 h of incubation without GH. No such decrease was seen in unfractionated adipocyte membrane preparations. The decrease in plasma membrane channel content was largely accounted for by redistribution of channels to a light microsomal membrane fraction. Immunoassay of alpha1-, alpha2/delta-, and beta-channel subunits in membrane fractions indicated that the channels redistributed as intact complexes. Addition of GH during the 1st h of incubation prevented channel redistribution, and addition of GH after 3 h restored channel distribution to the GH-replete state of freshly isolated adipocytes. The studies suggest that GH may regulate the abundance of Ca2+ channels in the adipocyte plasma membrane and thereby modulate sensitivity to signals, the expression of which is Ca2+ dependent.

Two isoforms of the GH receptor, the full-length receptor (GHRL) and a short isoform (GHRS) that lacks the transmembrane and intracellular domains of GHRL, have been analyzed in rat tissue extracts by Western blotting and immunoprecipitation. Although quantitative estimates of GHRS and GHRL based on coprecipitation of [125I]GH indicated similar amounts of both isoforms in tissue extracts, the 110 kDa band corresponding to GHRL was generally not detected on Western blots without enrichment by immunoprecipitation. Two bands with electrophoretic mobilities corresponding to 38 and 42 kDa were present in extracts prepared from liver, muscle, and adipocytes. Western blots of the GH binding protein in rat serum also revealed two bands, but these had electrophoretic mobilities corresponding to 44 and 52 kDa. After digestion by endoglycosidase F, a single band with an electrophoretic mobility corresponding to 31 kDa was detected in samples from adipocytes, liver or serum, indicating that GHRS retained in tissues is glycosylated less extensively than that in rat serum. Digestion with neuraminidase indicated that the smaller glycoproteins in tissue extracts lack sialic acid residues that are present in serum samples. Furthermore, endoglycosidase H degraded GHRS in liver extracts to a 31 kDa band but did not degrade serum samples, suggesting that tissues retain a high mannose form of GHRS. The abundance of GHRS or GHRL in tissues from male, virgin female, and pregnant rats was estimated from the amount of 125I-GH that was bound to each isoform after immunoprecipitation. Liver contained more than 10 times as much GHRS per gram of tissue as fat or muscle. In liver, muscle, and fat, the amount of GHRS exceeded that of GHRL, sometimes by as much as 6-fold. GHBP levels in serum of females exceeded those in males, and rose even higher in pregnant females. The abundance of GHRS in all tissue extracts paralleled serum levels. In muscle and fat, the levels of GHRL did not differ in male, female and pregnant rats, whereas in liver, the pattern was similar to the GHRS pattern. In all tissues, pools of GHRS exceeded those of GHRL by a factor that grew larger as tissue and serum levels increased. The half life of GHBP in serum was estimated to be 2.4 h in rats treated with cycloheximide, whereas that of GHRS was 20 min in liver and 8.5 h in fat. These results suggest that GHRS is synthesized in liver 8 times faster than it is released into serum, whereas synthesis in fat is less than 30% of the rate at which it is released into serum by all tissues. Therefore, liver appears to be the major source of GHBP in serum. Although secretion into the circulatory system accounts for little or perhaps none of its turnover in some tissues, GHRS pools in tissues do appear to be regulated, suggesting that GHRS may function primarily in the cells in which it is synthesized.

Arabidopsis abscisic acid (ABA)-insensitive abi4 mutants have pleiotropic defects in seed development, including decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression. This phenotype is consistent with a role for ABI4 in regulating seed responses to ABA and/or seed-specific signals. We isolated the ABI4 gene by positional cloning and confirmed its identity by complementation analysis. The predicted protein product shows homology to a plant-specific family of transcriptional regulators characterized by a conserved DNA binding domain, the APETALA 2 domain. The single mutant allele identified has a single base pair deletion, resulting in a frameshift that should disrupt the C-terminal half of the protein but leave the presumed DNA binding domain intact. Expression analyses showed that despite the seed-specific nature of the mutant phenotype, ABI4 expression is not seed specific.

Racemic albuterol has been one of the most widely used beta2-adrenoceptor agonists for the relief of the symptoms of asthma, yet the use of beta2 agonists has been known to induce bronchial hyperresponsiveness. To probe a possible role of the S-enantiomer for hyperresponsiveness, we determined the effects of (S)-albuterol on intracellular Ca2+ concentration ([Ca2+]i) in dissociated bovine tracheal smooth muscle cells. Both (S)-and (R,S)-albuterol increased [Ca2+]i at concentrations of >10 pM and 1 nM, respectively, with a maximal response by 150 and 100 nM, respectively. (S)-Albuterol (1 and 10 muM) induced Ca2+ oscillations, reaching 1-2 muM [Ca2+]i. This response is in a stark contrast to that of (R)-albuterol, which decreased [Ca2+]i. The increase in [Ca2+]i was blocked by 100 nM atropine or 500 nM 4-diphenylacetoxy-N-methylpiperidine but was insensitive to the beta2 antagonist ICI 118,551 (10 muM). (S)-Albuterol (10 muM) increased inositol-1,4,5-trisphosphate levels by 213 +/- 34.4% (p < 0.05, four experiments) in cells exposed for 30 sec. The sustained phase of the Ca2+ increase was absent in Ca2+-free solution, suggesting that Ca2+ influx was responsible for the sustained Ca2+ response. The results also suggest that (S)-albuterol may cross-react with muscarinic receptors. As a Ca2+ agonist in airway smooth muscle, (S)-albuterol may have profound clinical implications because 50% of prescribed racemic albuterol is composed of (S)-albuterol.

The function of Lhca4, a gene encoding the photosystem 1 type IV chlorophyll a/b-binding protein complex in Arabidopsis, was investigated using antisense technology. Lhca4 protein was reduced in a number of mutant lines and abolished in one. The inhibition of protein was not correlated with the inhibition of mRNA. No depletion of Lhca1 was observed, but the low-temperature fluorescence emission spectrum was drastically altered in the mutants. The emission maximum was blue-shifted by 6 nm, showing that chlorophyll molecules bound to Lhca4 are responsible for most of the long-wavelength fluorescence emission. Some mutants also showed an unexplainable delay in flowering time and an increase in seed weight.