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Results for “Pathway informed transformer”

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PubMed2026

Immune subtyping of colorectal adenoma identifies a subtype with activated adaptive immunity ahead of progressing to cancer.

BACKGROUND: Colorectal adenomas (CRA) represent precursor lesions with varying risks of malignant transformation. However, molecular subtyping, particularly immune-related classification, remains underexplored in adenomas. This study aims to characterize the immune landscape of CRA through immune subtyping and evaluate its association with cancer progression, gene expression signatures, and functional pathways. METHODS: We conducted a retrospective analysis of transcriptomic data from multiple cohorts of CRA samples. Immune subtypes were identified using non-negative matrix factorization (NMF) based on immune-related genes. Diverse deconvolution algorithms were used to estimate immune cell infiltration. The immune status alteration in premalignant lesion was further consolidated by single-cell transcriptome data. Differential gene expression analysis was performed between subtypes, followed by functional enrichment analyses (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]). RESULTS: Two distinct immune subtypes were identified: an immune-enriched subtype characterized by high lymphocyte infiltration and elevated expression of immune-related genes, and an immune-deficient subtype with suppressed immune activity. Differential expression analysis revealed significant upregulation of immune response genes (e.g., CD4, CD86, HLA-DRA) in the immune-enriched subtype. GO and KEGG analyses highlighted enrichments in leukocyte transendothelial migration, chemokine signaling, and antigen processing and presentation pathways. Single-cell result revealed an early occurrence of TIGIT activation and exhausted CD8 T cell features in adenoma when compared to normal tissue. CONCLUSION: This study delineates distinct immune subtypes within CRAs. The immune-enriched subtype demonstrates activated adaptive immunity and may reflect a higher potential for immune surveillance, while the immune-deficient subtype exhibits stromal features suggestive of progressive transformation. These findings provide insights into early immune microenvironment alterations and may inform strategies for risk stratification and immunoprevention in colorectal carcinogenesis.

Colorectal adenoma↗

PubMed2006

Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming.

Transgene expression from the chloroplast (plastid) genome offers several attractions to plant biotechnologists, including high-level accumulation of foreign proteins, transgene stacking in operons and a lack of epigenetic interference with the stability of transgene expression. In addition, the technology provides an environmentally benign method of plant genetic engineering, because plastids and their genetic information are maternally inherited in most crops and thus are largely excluded from pollen transmission. During the past few years, researchers in both the public and private sectors have begun to explore possible areas of application of plastid transformation in plant biotechnology as a viable alternative to conventional nuclear transgenic technologies. Recent proof-of-concept studies highlight the potential of plastid genome engineering for the expression of resistance traits, the production of biopharmaceuticals and metabolic pathway engineering in plants.

Agriculture↗

PubMed2005

A validated stability-indicating HPLC with photodiode array detector (PDA) method for the stress tests of Monascus purpureus-fermented rice, red yeast rice.

A stability-indicating reversed phase high-performance liquid chromatography (RP-HPLC) with photodiode array (PDA) detection method was developed and validated for the assay of monacolin series compounds including monacolin K, L, J and their hydroxyl acid forms as well as dehydroxymonacolin K simultaneously in Monascus purpureus-fermented rice, red yeast rice. Well-resolved peaks of seven main compounds of monacolin family were profiled on a C(18) reverse-phase column using a linear gradient of 0.1% trifluoroacetic acid and acetonitrile as the mobile phase, and the detection wavelength was set at 237nm. The method was validated with respect to specificity, chromatographic parameters, linearity, precision, accuracy, limits of detection and quantitation. The stability stress testing for fermented red yeast rice powder was carried out to show the effects of high temperature (80 degrees C), high humidity at room temperature (92.5% RH, 25 degrees C), high humidity at high temperature (75% RH, 60 degrees C) and light (sunlight) in solid state. The results exhibited that monacolins decreased significantly under the conditions of high humidity at high temperature (75% RH, 60 degrees C) and sunlight. Monacolin K and its hydroxyl acid form would be dehydrolyzed and turned to dehydromonacolin K at high temperature (80 degrees C) while the monacolin K, J and L would be transformed into their corresponding hydroxyl acid forms under the condition of high humidity (92.5% RH, 25 degrees C). The indication is that monacolins in red yeast rice powder are light-sensitive and thermal-sensitive. Therefore, it has been suggested that the preparations containing monacolins be stored in the place of cool and lightproof. The proposed degradation pathways were discussed as well. The multi-components assay for stability of botanical products could provide much more information than the normal marker-orientation method.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

The physicochemical and biological properties of alpha-fetoprotein depend of its ligand environment.

The majority of studies have concentrated on the endocrine and immunological roles of alpha-fetoprotein (AFP). These studies have been strongly orientated and sustained by research showing that rat and mouse AFPs competitively bind estrogen and non-esterified fatty acids. AFPs of other species, such as humans, are not estrogenophilic but all those studied to date bind NEFAs tightly. Endogenous ligands can greatly influence the conformation of the protein and consequently its biological activity. AFP may have three possible mechanisms of action: a) Murine AFP may be a modulatory element of estrogen expression which determines, under the control of NEFAs the concentration of the active free steroid. b) The protein could be transformed by bound ligands, so enhancing or inhibiting its binding to a target cell receptor, and consequently modifying its biological activity. AFP per se, can be the regulatory element. c) AFPs of all species can work as fatty acid binding proteins and modulate the availability of free NEFAs (not bound to protein). These free NEFAs can, in turn, act positively or negatively on various proteins involved in the pathways of the transfer of the steroid and peptide informations leading to cell multiplication or differentiation.

Animals↗

PubMed2005

TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation; interestingly its action is clearly cell type-dependent. In particular, it inhibits epithelial and endothelial cells' proliferation, while its action on many mesenchymal cells has been reported to be stimulatory. In this direction, we have recently shown that TGF-beta regulates the proliferation of normal human skin fibroblasts according to their developmental origin: i.e. it inhibits fetal fibroblasts, while it stimulates the proliferation of adult ones. Here, we present evidence on the mechanisms underlying this differential action. Concerning fetal fibroblasts, we have found that TGF-beta activates Protein Kinase A (PKA) and induces the expression of the cyclin-dependent kinase inhibitors (CKIs) p21(CIP1/WAF1) and p15(INK4B). Moreover, the specific PKA inhibitor H-89 blocks the induction of both CKIs and annuls the TGF-beta-mediated inhibitory effect, indicating the central role of PKA in this process. In contrast, in adult cells no PKA activation is observed. Moreover, TGF-beta stimulates cell proliferation by activating the MEK-ERK pathway, as the MEK inhibitor PD98059 blocks this effect. A specific neutralizing antibody against Fibroblast Growth Factor-2 (FGF-2) inhibits both ERK activation and the mitogenic activity of TGF-beta, indicating that the latter establishes an autocrine loop, via FGF-2, leading to cell proliferation. This loop requires FGF receptor-1 (FGFR-1), as its down-regulation by siRNA approach prevents TGF-beta from stimulating ERK-1/2 activation and DNA synthesis. In conclusion, the differential proliferative response of fetal and adult normal human skin fibroblasts to TGF-beta is regulated by distinct signaling pathways and furthermore it may provide information on the bimodal effect of this factor on cell proliferation, in general.

Adult↗

PubMed1995

The clonogenic precursor cell in multiple myeloma.

Multiple myeloma is characterized by the monoclonal expansion of plasma cells in the bone marrow. Although the predominant cell type is the plasma cell, the initial oncogenic transformation is considered to take place in a more immature B cell. There is still much controversy about this precursor cell type. Phenotypic analysis of bone marrow and peripheral blood revealed that in multiple myeloma a great diversity exists in the phenotype of the cells considered to be involved. Because of the lack of a myeloma specific genetic lesion it is very difficult to trace back the cell in which the transforming event, leading to multiple myeloma, took place. The only real clonal marker is the idiotype of the immunoglobulin molecule expressed by the myeloma cells. With recombinant DNA technology it is now possible to produce clonal markers for each individual myeloma patient which recognize only the immunoglobulin genes expressed by the myeloma cell and its precursors. The sequences of these myeloma immunoglobulin genes do reveal a lot of information about the stage in the B-cell differentiation pathway in which the oncogenic event might have taken place. The presence of somatic mutations in a non-random fashion without intraclonal variation leads to the conclusion that the precursor myeloma cell could not possibly be a pre-B cell or stem cell but has to be a mature B cell that has been in contact with antigen and has past through the phase of somatic mutation, like a memory B cell or plasmablast.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗

PubMed1991

Genetic competence in Bacillus subtilis.

Genetic competence may be defined as a physiological state enabling a bacterial culture to bind and take up high-molecular-weight exogenous DNA (transformation). In Bacillus subtilis, competence develops postexponentially and only in certain media. In addition, only a minority of the cells in a competent culture become competent, and these are physiologically distinct. Thus, competence is subject to three regulatory modalities: growth stage specific, nutritionally responsive, and cell type specific. This review summarizes the present state of knowledge concerning competence in B. subtilis. The study of genes required for transformability has permitted their classification into two broad categories. Late competence genes are expressed under competence control and specify products required for the binding, uptake, and processing of transforming DNA. Regulatory genes specify products that are needed for the expression of the late genes. Several of the late competence gene products have been shown to be membrane localized, and others are predicted to be membrane associated on the basis of amino acid sequence data. Several of these predicted protein sequences show a striking resemblance to gene products that are involved in the export and/or assembly of extracellular proteins and structures in gram-negative organisms. This observation is consistent with the idea that the late products are directly involved in transport of DNA and is equally consistent with the notion that they play a morphogenetic role in the assembly of a transport apparatus. The competence regulatory apparatus constitutes an elaborate signal transduction system that senses and interprets environmental information and passes this information to the competence-specific transcriptional machinery. Many of the regulatory gene products have been identified and partially characterized, and their interactions have been studied genetically and in some cases biochemically as well. These include several histidine kinase and response regulator members of the bacterial two-component signal transduction machinery, as well as a number of known transcriptionally active proteins. Results of genetic studies are consistent with the notion that the regulatory proteins interact in a hierarchical way to make up a regulatory pathway, and it is possible to propose a provisional scheme for the organization of this pathway. It is remarkable that almost all of the regulatory gene products appear to play roles in the control of various forms of postexponential expression in addition to competence, e.g., sporulation, degradative-enzyme production, motility, and antibiotic production. This has led to the notion of a signal transduction network which transduces environmental information to determine the levels and timing of expression of the ultimate products characteristic of each of these systems.

Amino Acid Sequence↗

PubMed2006

Role of alterations in the apoptotic machinery in sensitivity of cancer cells to treatment.

Apoptosis is a genetically controlled and evolutionarily conserved form of cell death of critical importance for normal embryonic development and for the maintenance of tissue homeostasis in the adult organism. The malfunction of the death machinery may play a primary or secondary role in various diseases, with essentially too little or too much apoptosis leading to proliferative or degenerative diseases, respectively. The machinery responsible for killing and degradation of the cell via apoptosis is expressed constitutively and become activated through various stimuli. Apoptotic mechanisms are operating during spontaneous regression of tumors as well as in response to treatment with anti-neoplastic drugs. The therapeutic goal in cancer treatment is to trigger tumor-selective cell death. However, resistance to treatment is a concern for many types of cancer. Since many anti-neoplastic agents induce an apoptotic type of death in susceptible cells, it is likely that defects or dysregulation of different steps of the apoptotic pathways might be an important determinant of resistance to anticancer drugs. These defects might appear at the initiation and/or execution stages of apoptosis and result in the insufficient elimination of tumor cells, which might lead either to acquired resistance to treatment, or to uncontrolled migration of cancer cells and metastasis. Hence, identification and targeting of the disabled pathway, which is most efficiently inactivated in a particular type of tumor might be the most successful approach in the future. Here we review current knowledge concerning function of apoptotic machinery in cancer cells, and how this information can be used to increase the efficiency of tumor treatment.

Animals↗

PubMed2001

Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection.

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.

Animals↗

PubMed2002

Cumulative bondomers: a new concept in flux analysis from 2D [13C,1H] COSY NMR data.

A well-established way of determining metabolic fluxes is to measure 2D [(13)C,(1)H] COSY NMR spectra of components of biomass grown on uniformly (13)C-labeled carbon sources. When using the entire set of measured data to simultaneously determine all fluxes in a proposed metabolic network model, the (13)C-labeling distribution in all measured compounds has to be simulated. This requires very large sets of isotopomer or cumomer balances. This article introduces the new concept of bondomers; entities that only vary in the numbers and positions of C-C bonds that have remained intact since the medium substrate molecule entered the metabolism. Bondomers are shown to have many analogies to isotopomers. One of these is that bondomers can be transformed to cumulative bondomers, just like isotopomers can be transformed to cumomers. Similarly to cumomers, cumulative bondomers allow an analytical solution of the entire set of balances describing a metabolic network. The main difference is that cumulative bondomer models are considerably smaller than corresponding cumomer models. This saves computational time, allows easier identifiability analysis, and yields new insights in the information content of 2D [(13)C,(1)H] COSY NMR data. We illustrate the theoretical concepts by means of a realistic example of the glycolytic and pentose phosphate pathways. The combinations of 2D [(13)C,(1)H] COSY NMR data that allow identification of all metabolic fluxes in these pathways are analyzed, and it is found that the NMR data contain less information than was previously expected.

Algorithms↗

PubMed2026

Open and sustainable AI: challenges, opportunities and the road ahead in the life sciences.

Artificial intelligence (AI) has seen transformative breakthroughs in the life sciences, expanding possibilities to interpret biological information at an unprecedented capacity. To maximize return on growing investments and accelerate progress, it is urgent to address long-standing research challenges arising from the rapid adoption of AI methods. We review the erosion of trust in AI outputs driven by poor reusability and reproducibility, and highlight their impact on environmental sustainability. Furthermore, we discuss the fragmented components of the AI ecosystem and lack of guiding pathways to support open and sustainable AI model development. In response, this Perspective introduces practical open and sustainable AI recommendations mapped to over 300 ecosystem components and provides guiding implementation pathways. Our work connects researchers with relevant AI resources, facilitating the implementation of sustainable, reusable and reproducible AI. Built upon community consensus and aligned to existing efforts, these outputs will aid future policy development and structured pathways for guiding AI implementation.

Artificial Intelligence↗

PubMed2007

15-Deoxy-Delta12,14-prostaglandin J2 enhances docetaxel anti-tumor activity against A549 and H460 non-small-cell lung cancer cell lines and xenograft tumors.

15-Deoxy-Delta-prostaglandin J2 is a naturally occurring endogenous ligand for peroxisome proliferator-activated receptor-gamma. The current study was aimed to determine the mechanism of anti-proliferative effect of 15-deoxy-Delta-prostaglandin J2+docetaxel against A549 and H460 non-small-cell lung cancer cell lines and xenograft tumors. In-vitro cytotoxicity of 15-deoxy-Delta-prostaglandin J2 alone and in combination with docetaxel was studied against A549 and H460 cell lines. For in-vivo studies, female athymic nu/nu mice were xenografted with A549 and H460 tumors and treated with 15-deoxy-Delta-prostaglandin J2 (1 mg/kg/day; intraperitoneal), docetaxel (10 mg/kg; intravenous on days 14, 18 and 22) and 15-deoxy-Delta-prostaglandin J2+docetaxel. Apoptosis was measured in A549 cells and tumor tissues, following various treatments. Peroxisome proliferator-activated receptor-gamma, caspases, Bcl2 and p53 family proteins or their mRNA expressions were measured by Western blotting, reverse transcription-polymerase chain reaction and real-time polymerase chain reaction in A549 tumors. A possible role of a peroxisome proliferator-activated receptor-gamma-independent mechanism was studied in A549 cells treated with peroxisome proliferator-activated receptor-gamma antagonist, GW9662. Isobolographic analysis demonstrated synergistic interaction (combination index <1.0) between 15-deoxy-Delta-prostaglandin J2 and docetaxel against A549 and H460 cells in vitro. 15-Deoxy-Delta-prostaglandin J2+docetaxel significantly reduced the tumor volume compared with control (P<0.05), 15-deoxy-Delta-prostaglandin J2 (P<0.05) and docetaxel (P<0.05, P<0.01) in both A549 and H460 tumors. 15-Deoxy-Delta-prostaglandin J2+docetaxel showed a significant increase in apoptosis associated with inhibition of the Bcl2 and cyclin D1 expression and overexpression of caspase and p53 pathway genes. Further, enhanced expression of caspase 3 and inhibition of cyclin D1 by 15-deoxy-Delta-prostaglandin J2+docetaxel was not reversed by GW9662, thus suggesting a possible peroxisome proliferator-activated receptor-gamma-independent mechanism. In conclusion, 15-deoxy-Delta-prostaglandin J2 enhanced the anti-tumor action of docetaxel by peroxisome proliferator-activated receptor-gamma-dependent and -independent mechanisms mediated by induction of apoptosis.

Animals↗

PubMed1990

Comprehensive two-dimensional gel protein databases offer a global approach to the analysis of human cells: the transformed amnion cells (AMA) master database and its link to genome DNA sequence data.

A total of 3430 polypeptides (2592 cellular; 838 secreted) from transformed human amnion cells (AMA) labeled with [35S]methionine were separated and recorded using computer-aided two-dimensional (2-D) gel electrophoresis. A master 2-D gel database of cellular protein information that includes both qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988, 2,561-602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity recovered from the gels (based on quantitations of polypeptides labeled with a mixture of 16 14C-amino acids), protein name (including credit to investigators that aided identification), antibody against protein, cellular localization, (nuclear, 40S hnRNP, 20S snRNP U5, proteasomes, endoplasmic reticulum, mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle-regulated proteins, proteins sensitive to interferons alpha, beta, and gamma, heat shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype-specific proteins, to microsequence them and store the information in the database, to identify the corresponding genes, to search for homology with previously characterized proteins and to study the function of groups of proteins (pathways, organelles, etc.) that exhibit interesting regulatory properties. In particular, the 2-D gel protein database may become increasingly important in view of the concerted effort to map and sequence the entire human genome.

Amnion↗

PubMed1990

Vitellogenesis in Drosophila: sequestration of a yolk polypeptide/invertase fusion protein into developing oocytes.

The mechanism of yolk deposition into developing oocytes of Drosophila was investigated by following the fate of a reporter protein fused to a vitellogenin, or yolk polypeptide (YP). Embryos were transformed with a hybrid gene consisting of the promotor and amino terminal 430 codons of the Yp2 gene fused to the cytoplasmic form of the invertase gene from the yeast Saccharomyces cerevisiae. RNA hybridization experiments with established lines of transformed flies showed that the hybrid gene was expressed in female fat bodies and ovaries but not in any male cells. Immunoblotting and endoglycosidase digestion showed that the hybrid protein was secreted from fat body cells via the secretory pathway, transported in hemolymph, and sequestered into developing oocytes. Transfusion experiments with hemolymph and pure invertase showed that sequestration of invertase depended on its attachment to YP. Immunocytochemistry demonstrated that the hybrid protein became localized in yolk granules as oocytes developed. Females homozygous for the fusion gene are generally sterile; their eggs containing the hybrid protein often collapse and their embryos fail to develop, suggesting that the structure of the yolk polypeptides is important for embryonic development. These experiments show that YP2 carries structural information sufficient to direct a reporter protein from fat body cells, through the hemolymph, and into the yolk granules of developing oocytes. This work provides a means of identifying the features of yolk polypeptides that are responsible for their deposition into yolk during oogenesis.

Amino Acid Sequence↗

PubMed2005

Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.

One of the major modes of activity of high-density cultures of dissociated neurons is globally synchronized bursting. Unlike in vivo, neuronal ensembles in culture maintain activity patterns dominated by global bursts for the lifetime of the culture (up to 2 years). We hypothesize that persistence of bursting is caused by a lack of input from other brain areas. To study this hypothesis, we grew small but dense monolayer cultures of cortical neurons and glia from rat embryos on multi-electrode arrays and used electrical stimulation to substitute for afferents. We quantified the burstiness of the firing of the cultures in spontaneous activity and during several stimulation protocols. Although slow stimulation through individual electrodes increased burstiness as a result of burst entrainment, rapid stimulation reduced burstiness. Distributing stimuli across several electrodes, as well as continuously fine-tuning stimulus strength with closed-loop feedback, greatly enhanced burst control. We conclude that externally applied electrical stimulation can substitute for natural inputs to cortical neuronal ensembles in transforming burst-dominated activity to dispersed spiking, more reminiscent of the awake cortex in vivo. This nonpharmacological method of controlling bursts will be a critical tool for exploring the information processing capacities of neuronal ensembles in vitro and has potential applications for the treatment of epilepsy.

Action Potentials↗

PubMed1995

Regulation by tumour antigens defines a role for PP2A in signal transduction.

Research conducted over the last decade has provided a wealth of new information on the molecular mechanisms utilized by DNA tumour viruses. Studies of tumour viruses have led to important insights into the functions of viral proteins and into the regulation of normal cellular proliferation. DNA tumour viruses can stimulate growth factor signaling pathways, alter gene transcription, and inactivate growth suppressor proteins. Members of the polyoma and adenovirus families express proteins that interact with protein serine/threonine phosphatase 2A (PP2A). In the case of SV40 virus, this interaction plays an accessory role in transformation of most cells, while it is essential for transformation of some cell types. The topics discussed in this review include the interactions of these viral proteins with PP2A, the effects of these interactions on phosphatase activity, and how these interactions alter cellular signal transduction pathways.

Amino Acid Sequence↗

PubMed2002

Chloroplast quest: a journey from the cytosol into the chloroplast and beyond.

Chloroplasts are characteristic organelles of plants and algae and the site of oxygenic photosynthesis. They are surrounded by a double membrane and possess an internal membrane system, the thylakoids, on which the photosynthetic machinery is located. They originated more than 1.2 billion years ago from an endosymbiotic event between an already photosynthetic ancestor of present day cyanobacteria and a mitochondriate host cell. During the transformation of the internalized cyanobacterium into a cell organelle most of the genetic information of the endosymbiot got lost or was transferred into the nucleus of the host. Chloroplast proteins encoded by nuclear genes are synthesized on cytoplasmic ribosomes and have to be relocated into the organelle. This is achieved by a proteinaceous import machinery in the outer and inner envelope of the chloroplasts. Proteins destined for the thylakoid membrane and the thylakoid lumen are further translocated by several different pathways into or across this membrane. The subject of this review is the quest of nuclear encoded chloroplast proteins into the organelle and to their final suborganellar location.

Arabidopsis↗

PubMed2002

RhoGEF specificity mutants implicate RhoA as a target for Dbs transforming activity.

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. Like many RhoGEFs, the in vitro catalytic activity of Dbs is not limited to a single substrate. It can catalyze the exchange of GDP for GTP on RhoA and Cdc42, both of which are expressed in most cell types. This lack of substrate specificity, which is relatively common among members of the RhoGEF family, complicates efforts to determine the molecular basis of their transforming activity. We have recently determined crystal structures of several RhoGEFs bound to their cognate GTPases and have used these complexes to predict structural determinants dictating the specificities of coupling between RhoGEFs and GTPases. Guided by this information, we mutated Dbs to alter significantly its relative exchange activity for RhoA versus Cdc42 and show that the transformation potential of Dbs correlates with exchange on RhoA but not Cdc42. Supporting this conclusion, oncogenic Dbs activates endogenous RhoA but not endogenous Cdc42 in NIH 3T3 cells. Similarly, a competitive inhibitor that blocks RhoA activation also blocks Dbs-mediated transformation. In conclusion, this study highlights the usefulness of specificity mutants of RhoGEFs as tools to genetically dissect the multiple signaling pathways potentially activated by overexpressed or oncogenic RhoGEFs. These ideas are exemplified for Dbs, which is strongly implicated in the transformation of NIH 3T3 cells via RhoA and not Cdc42.

3T3 Cells↗