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PubMed1999

Deafferentation and pointing with visual double-step perturbations.

The capability of reprogramming movement responses following changes in the visual goal has been studied through the double-step paradigm. These studies have shown that: (a) continuous internal feedback-loops correct unconsciously the dynamic errors throughout the movement; (b) proprioceptive information and/or the efference copy have a privileged status among central processes, insuring on-line regulation of the initial motor commands; and (c) generation of the motor program starts after target presentation, and is continuously updated in the direction of the current internal representation of the target, at least until the onset of hand movement. This main corrective process of the initial program appears to be basically independent of visual reafference from the moving hand. However, the agreement with the possibility of a visuomotor loop, based on the comparison of the new updated representation of the target position and on the information from the moving hand, has not determined whether the correcting process is proprioceptive feedback dependent, or whether internal feedback-loops (efferent copies) are responsible for quick corrections of unfolding motor responses. To answer this question, the present experiment investigated the pointing behavior of a deafferented subject, using a double-step paradigm under various conditions of visual feedback and movement initiation. Overall, the present study (a) clearly showed the capacity of the motor system to modify and correct erroneous trajectories on the mere basis of internal feedback-loops and (b) emphasized the crucial role played by the target jump/arm triggering delay and the importance of the eye efferent copy for providing information about the spatial goal of the movement.

Afferent Pathways↗

PubMed2006

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of sugars, alcohols, and organic acids with transcriptional microarrays.

Microorganisms respond to environmental changes by reprogramming their metabolism primarily through altered patterns of gene expression. DNA microarrays provide a tool for exploiting microorganisms as living sensors of their environment. The potential of DNA microarrays to reflect availability of nutrient components during fermentations on complex media was examined by monitoring global gene expression throughout batch cultivation of Escherichia coli MG1655 on Luria-Bertani (LB) medium. Gene expression profiles group into pathways that clearly demonstrate the metabolic changes occurring in the course of fermentation. Functional analysis of the gene expression related to metabolism of sugars, alcohols, and organic acids revealed that E. coli growing on LB medium switches from a sequential mode of substrate utilization to the simultaneous one in the course of the growth. Maltose and maltodextrins are the first of these substrates to support growth. Utilization of these nutrients associated with the highest growth rate of the culture was followed by simultaneous induction of enzymes involved in assimilation of a large group of other carbon sources including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol, amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of these nutrients to the cells was monitored by induction of corresponding transport and/or catabolic systems specific for each of the compounds.

Carbohydrate Metabolism↗

PubMed2025

Integrating necroptosis and immune landscapes: a multi-omics-derived NecropImmScore stratifies prognosis and therapy in ovarian cancer.

BACKGROUND: Ovarian cancer (OC) remains the deadliest gynecologic malignancy, largely due to its immunosuppressive tumor microenvironment (TME) and resistance to therapy. Necroptosis, a regulated lytic cell death pathway mediated by the RIPK1-RIPK3-MLKL axis, can trigger immunogenic cell death, but its specific role in shaping the OC immune landscape and its clinical translation potential are posorly understood. METHODS: We employed multi-omics analysis (transcriptomics, genomics, clinical data) from TCGA-OV (n = 380), ICGC OV-AU, and IMvigor210 cohorts, combined with rigorous in vitro functional validation using OC cell lines (SKOV3, HEY), macrophages (THP-1 derived), and T cells (Jurkat). Computational immunology approaches (ESTIMATE, CIBERSORT, ssGSEA) quantified immune infiltration. We identified MLKL-associated immune genes, performed survival analysis (Kaplan-Meier, Cox regression), and constructed a necroptosis-immune signature (NecropImmScore) using consensus clustering and PCA of 102 prognostic genes. Drug sensitivity was predicted via pRRophetic and CellMiner. RESULTS: MLKL emerged as a protective prognostic biomarker (p = 0.018), significantly correlated with enhanced immune infiltration (ImmuneScore, StromalScore, ESTIMATEScore; p < 2.22e-16), M1 macrophage polarization (p = 0.006), activated CD4 + T cells (p = 0.003), and elevated immune checkpoint expression (PD-L1, CTLA4, LAG3, TIGIT). In vitro, MLKL overexpression in OC cells promoted M1 polarization (p < 0.05), activated Jurkat T cells (upregulated CCR4/5/7/9, CD69, CD3D/E, GZMB; p < 0.05), and induced key chemokines (CXCL9/10/11/13) critical for immune cell recruitment. Integration of MLKL-related and immune-related DEGs (n = 632) revealed enrichment in T-cell activation, chemokine signaling, and antigen presentation pathways (FDR < 0.05). Consensus clustering based on 102 survival-associated genes defined three molecular subtypes (Clusters A-C) with divergent survival (p = 0.019), necroptosis activity, and immune infiltration (Cluster C: best prognosis, highest MLKL/ImmuneScore). The derived NecropImmScore robustly stratified patients: high-score correlated with superior overall survival (TCGA: p < 0.001; ICGC: p = 0.014), inflamed TME phenotype, elevated checkpoint expression, and improved response to anti-PD-L1 in IMvigor210. Critically, high NecropImmScore predicted higher BRCA1 mutation frequency (AUC = 0.802), synergy with BRCA1 status for prognosis, higher homologous recombination deficiency (HRD) score, sensitivity to cisplatin (p = 0.014), paclitaxel (p = 0.016), gemcitabine (p = 0.017), and provided superior prognostic stratification when combined with TMB and HRD score (p < 0.001). CONCLUSION: This study establishes MLKL as a master regulator of anti-tumor immunity in OC, driving chemokine-mediated immune cell recruitment and TME reprogramming. The novel NecropImmScore is a multifaceted biomarker that effectively predicts prognosis, immunotherapy response, BRCA1 deficiency, and chemosensitivity, offering significant potential for guiding precision therapeutic strategies in OC.

Humans↗

PubMed2006

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment.

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25 degrees C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25-27 degrees C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.

Embryonic Development↗

PubMed2026

Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding.

This review synthesizes the molecular mechanisms of cold tolerance in pepper, integrating multi-omics data,genome editing, and precision breeding strategies to accelerate the development of cold-resilient cultivars. Cold stress is a significant environmental factor that affects the growth, productivity, and fruit quality of Capsicum annuum by impairing membrane integrity photosynthesis and cellular redox homeostasis. Although pepper has several endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, its cold tolerance remains limited due to low transcriptional activation of key regulators, functional redundancy among cold-responsive genes, and the polygenicity of cold tolerance. These complexities, combined with low genetic diversity and linkage drag, have hindered the improvement of cold-resistant cultivars through conventional breeding. This review brings together the recent progress in understanding the molecular mechanisms of cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Precision Breeding 2.0 is a new innovation that combines the integration of multi-omics-based target identification with next-generation genome-editing techniques, allowing precise and multiplex engineering of complex and interconnected regulatory networks instead of single genes. We cover new approaches such as engineering the DREB/CBF pathway, allele-specific editing and targeted disruption of negative regulators to enhance the pathway(s) involved in cold response. Moreover, we propose a roadmap for integration of transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to accelerate the identification, validation, and deployment of superior alleles to boost cold tolerance. This review provides a foundation for developing climate-resilient pepper cultivars by connecting functional genomics with precision genome engineering approaches to maintain productivity under variable environmental conditions.

Capsicum↗

PubMed2005

Seasonal modulation of growth hormone mRNA and protein levels in carp pituitary: evidence for two expressed genes.

Adaptation of eurythermal fish to naturally varying environmental conditions involves modulation of expressions of various factors in the hypothalamo-hypophyseal axis. Here we used three complementary approaches to assess the seasonal variation of growth hormone (GH) protein and mRNA levels in pituitary glands of acclimatized carp fish. First, a polyclonal antibody raised against an oligopeptide derived from the carp GH sequence was used for immunohistochemistry; second, oligonucleotides specific for GH transcripts were used for in situ hybridization. Specific immunodetection of GH coincides with visualization of GH mRNA in the proximal pars distalis, the specific location of somatotroph cells in carp pituitary gland. Finally, competitive RT-PCR analyses confirmed that GH expression exhibits seasonal cyclical reprogramming with higher levels in summer- than in winter-adapted fish. The expression pattern suggests an important role for GH in the molecular mechanisms underlying the acclimatization process. In parallel, amplification of sequences from the fourth intron and adjacent sites from exons IV and V demonstrates the existence of a new GH gene previously undescribed. The detection of transcripts corresponding to each gene suggests that both GH gene copies are active in the duplicated carp genome and that they are similarly affected by seasonal adaptation.

Acclimatization↗

PubMed2002

[Surgical and interventional use of radiofrequency current: is there interference with implantable cardioverter/defibrillators?].

BACKGROUND: During surgical and interventional procedures, interactions between implantable cardioverter defibrillators (ICD) and electrical cautery, respectively, application of radiofrequency (RF) energy may occur. Induction of inadequate shock therapies or device malfunction may result and represent a potential perioperative hazard for the patient. METHODS: Hence, we analyzed the intraoperative interactions in 23 consecutive ICD patients with regard to different surgical and interventional procedures. Sixteen surgical operations (general surgery n = 7, urologic n = 5, abdominal n = 2, gynecological n = 1, thoracic n = 1) and 7 interventional therapies (RF catheter ablation n = 5, endoscopic papillotomy n = 2) were performed. The ICD devices were all located in the left pectoral position and consisted of 15 single and 8 dual chamber defibrillators. During the procedure tachyarrhythmia detection (VF 295 +/- 21 ms, VT 370 +/- 55 ms) of the devices was maintained active (monitoring mode); only ICD therapies were inactivated. The indifferent electrode of the electrical cauter/RF generator was placed in standard positions (right mid femoral position n = 18, thoracic spine area n = 5). After the procedure, the ICD memory was checked for detections, respectively, for changes of the programming. RESULT: There was no misdetection or reprogramming of the ICD caused by electrical cautery or RF energy. CONCLUSIONS: Despite the lack of undesired interactions ICDs should be inactivated preoperatively to assure maximum patient safety. However, should inactivation be ineffective or not manageable, electromagnetic interference is highly unlikely.

Adult↗

PubMed2003

Cardiac rehabilitation in patients with implantable defibrillators. Feasibility and complications.

OBJECTIVE: We examined patients with implantable cardioverter defibrillators (ICD) in order to demonstrate their safe participation in a standard rehabilitation program. DESIGN: Prospective cohort study of a consecutive series of patients after ICD implantation. Setting Inpatient rehabilitation center. PATIENTS AND METHODS: A total of 118 patients (73.7% male, mean age 60+/-11 years) took part for 23 +/- 4 days in a standard inpatient rehabilitation program including physical activity, psychological care, heart function seminars, and resuscitation exercises with family members. The following noninvasive tests were performed: symptomlimited exercise testing, two-dimensional echocardiography, Holter monitoring, telemetric ICD interrogation, optional fluoroscopy or X-ray examination of the thorax, and (in some patients) defibrillation threshold testing. RESULTS: Out of 118 patients 101 patients (85.6%) participated in regular ergometer training during which the initial workload of 23 +/- 11 Watts could be increased to 45 +/- 18 Watts. An individual conditioning program was assigned to 15% (n = 17) patients, thereby enabling the inclusion of all patients in the rehabilitation process. Under these conditions 12 patients (10%) experienced ICD malfunctions requiring therapy. As a consequence of all cardiac function tests, ICD reprogramming was necessary in 26 patients (22.1%). CONCLUSION: Following ICD implantation, patients may participate in a standard rehabilitation program without serious complications and with a significant increase in physical capacity. However, ICD malfunction occurs in approximately 10% of patients. Additional tests performed by skilled medical staff and appropriate technical equipment allows the ICD program to be optimized.

Aged↗

PubMed1997

[Combined therapy with transvenous cardioverter/defibrillator and anti-bradycardia pacemaker systems].

Implantable cardioverter/ defibrillators (ICD) represent in many patients with ventricular tachyarrhythmias the first line treatment. Up to 15% of the patients requiring an ICD need concomitant permanent cardiac pacing for bradyarrhythmias resulting in the need of simultaneous ICD- and antibradycardic pacemaker-therapy. We present four patients with successful implantation of a transvenous ICD-system (Medtronic Jewel 7220 and Micro Jewel 7221; electrode: Medtronic 6939), all of which had an antibradycardic pacemaker (2 unipolar, 2 bipolar) implanted prior to ICD-implantation. Meticulous intraoperative testing in order to avoid adverse interactions between the two systems has been carried out successfully in all cases. Possible adverse interactions comprise oversensing of pacemaker signals by the ICD with subsequent inadequate therapy, inhibition of defibrillation therapy during ventricular fibrillation caused by false detection of pacemaker spikes by the ICD as well as reprogramming of the pacemaker after shock delivery. A review of the published literature yielded 91 reported cases of combined ICD- and pacemaker-therapy. In 16% of the patients, one or more of the mentioned complications have been observed. Those occurred more frequently with unipolar than with bipolar pacemaker-systems. Thus, combined therapy with antibradycardic pacemaker and transvenous ICD-systems can be performed safely.

Aged↗

PubMed2003

Chromosomal proteins in the spermatogenesis of Drosophila.

Chromatin constitution in the male germ line of Drosophila is discussed with respect to the substitution of somatic histones by protamines or other basic proteins. The specific properties of germ line chromatin include the initiation and completion of the spermatogenic pathway and the reprogramming of the genome for embryonic development. During meiotic prophase cell cycle-regulated H3 histones appear to a large extent to be substituted by the histone H3.3 replacement variant protein, which is generally found associated with transcriptionally active chromatin. Condensation of the chromosomes during meiosis and the subsequent compaction for packaging in the sperm head require suitable proteins, but the cell cycle-regulated histones are not available as their expression is limited to S-phase. It is, therefore, proposed that any basic protein with a limited range of sequence requirements may take over this packaging function. Suitable proteins may have evolved by divergence from histone variants not restricted in their expression to S-phase, similar to the testes-predominant histone H3.3A of Drosophila.

Animals↗

PubMed2005

Developmental regulation of Suz 12 localization.

Chromatin modifications are among the epigenetic alterations essential for genetic reprogramming during development. The Polycomb group (PcG) gene family mediates chromatin modifications that contribute to developmentally regulated transcriptional silencing. Trimethylation of histone H3 on lysine 27, mediated by a PcG protein complex consisting of Eed, Ezh2, and Suz12, is integral in differentiation, stem cell self-renewal, and tumorigenesis. Eed and Ezh2 are also implicated in the developmentally regulated silencing of the inactive X chromosome, as they are transiently enriched on the inactive X chromosome when X chromosome silencing is initiated. Here we analyze the dynamic localization of Suz12 during cellular differentiation and X-inactivation. Though Suz12 is a requisite member of the Eed/Ezh2 complexes, we found that Suz12 exhibits a notable difference from Ezh2 and Eed: while Ezh2 and Eed levels decrease during stem cell differentiation, Suz12 levels remain constant. Despite the differential regulation in abundance of Suz12 and Eed/Ezh2, Suz12 is also transiently enriched on the Xi during early stages of X-inactivation, and this accumulation is Xist RNA dependent. These results suggest that Suz12 may have a function that is not mediated by its association with Eed and Ezh2, and that this additional function is not involved in the regulation of X-inactivation.

Animals↗

PubMed2004

Nucleoskeleton of early bovine embryos and differentiated somatic cells: an ultrastructural and immunocytochemical comparison.

The nucleoskeleton is a complex structure involved in structural and functional organisation of the genome in eukaryotic cells. As little information on the nucleoskeleton is available from early embryonic development stages, we describe here the morphology and composition of the nucleoskeleton in cleaving bovine embryos (stages 1-16 cells). Ultrastructural observations were performed using thick resinless sections after chromatin removal by nucleases. The localisation of nucleoskeleton-related lamins A and C, NuMA, SRm160 and hnRNP H was tested by immunofluorescence. The characteristic structures of the nucleoskeleton (nuclear lamina, core filaments and the 'diffuse' nucleoskeleton) were present throughout all embryonic stages studied, although less discernable during the 1-cell stage. Lamins A and C as well as the NuMA protein were observed in embryo nuclei from the 1-cell stage; a diffuse hnRNP H and speckled SRm160 immunofluorescence appeared from the 4-cell stage. During the 8- to 16-cell stages (major transcriptional activation), the immunofluorescence patterns were identical with those of differentiated cells (fibroblasts). The temporal pattern of immunolabelling confirmed that transcription and splicing compartmentalisation was established progressively during cleavage, and that some of the proteins tested can be used as markers in studies on nuclei reprogramming after transfer into enucleated oocytes.

Animals↗

PubMed2003

A comparison of early floral ontogeny in wild-type and floral homeotic mutant phenotypes of Primula.

Primula flowers are heteromorphic with individual plants producing either pin-form or thrum-form flowers. We have used scanning electron microscopy to observe early development of wild-type flowers of primrose (Primula vulgaris), cowslip (P. veris), and the polyanthus hybrid (P. x tommasinii x P. vulgaris). Floral ontogeny in Primula is different from that observed in the well-studied models Antirrhinum majus and Arabidopsis thaliana and our studies reveal morphological landmark events that define the sequence of early floral development in Primula into specific stages. Pin-form and thrum-form flowers are indistinguishable during early development with differentiation of the two floral morphs occurring beyond the differentiation of floral organs. Early ontogeny of flowers with homeotic mutant phenotypes was also studied to determine the timing of developmental reprogramming in these mutants. Phenotypes studied included Hose in Hose and Jack in the Green that develop petaloid sepals and leafy sepals, respectively, and Jackanapes plants that carry both these dominant mutations. Recessive double and semi- double flowers that produce additional whorls of petals and/or stamens in place of carpels were also studied. We describe a previously undocumented recessive Primula mutant phenotype, sepaloid, that produces sepals in place of petals and stamens, and a new non-homeotic, dominant mutant phenotype Split Perianth, in which sepals and petals fail to fuse to form the typical calyx and corolla structures. The molecular basis of these mutant phenotypes in relation to the ABC model is discussed.

Flowers↗

PubMed1998

Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.).

Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the invitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277-1289; N. Sinha et al., 1993 Genes Dev. 7: 787-797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro.

Antibodies↗

PubMed2005

Genome-wide mRNA profiling in glucose starved Bacillus subtilis cells.

In this study global changes in gene expression were monitored in Bacillus subtilis cells entering stationary growth phase owing to starvation for glucose. Gene expression was analysed in growing and starving cells at different time points by full-genome mRNA profiling using DNA macroarrays. During the transition to stationary phase we observed extensive reprogramming of gene expression, with approximately 1,000 genes being strongly repressed and approximately 900 strongly up-regulated in a time-dependent manner. The genes involved in the response to glucose starvation can be assigned to two main classes: (i) general stress/starvation genes which respond to various stress or starvation stimuli, and (ii) genes that respond specifically to starvation for glucose. The first class includes members of the sigma(B)-dependent general stress regulon, as well as 90 vegetative genes, which are strongly down regulated in the course of the stringent response. Among the genes in the second class, we observed a decrease in the expression of genes encoding proteins required for glucose uptake, glycolysis and the tricarboxylic acid cycle. Conversely, many carbohydrate utilisation systems that depend on phosphotransferase systems (PTS) or ABC transporters were activated. The expression of genes required for utilisation or generation of acetate indicates that acetate constitutes an important energy source for B. subtilis during periods of glucose starvation. Finally, genome wide mRNA profiling data can be used to predict new metabolic pathways in B. subtilis. Thus, our data suggest that glucose-starved cells are able to degrade branched-chain fatty acids to pyruvate and succinate via propionyl-CoA using the methylcitrate pathway. This pathway appears to link lipid degradation to gluconeogenesis in glucose-starved cells.

Bacillus subtilis↗

PubMed2006

The transcription factor Gcr1 stimulates cell growth by participating in nutrient-responsive gene expression on a global level.

Transcriptomic reprogramming is critical to the coordination between growth and cell cycle progression in response to changing extracellular conditions. In Saccharomyces cerevisiae, the transcription factor Gcr1 contributes to this coordination by supporting maximum expression of G1 cyclins in addition to regulating both glucose-induced and glucose-repressed genes. We report here the comprehensive genome-wide expression profiling of gcr1Delta cells. Our data show that reduced expression of ribosomal protein genes in gcr1Delta cells is detectable both 20 min after glucose addition and in steady-state cultures of raffinose-grown cells, showing that this defect is not the result of slow growth or growth on a repressing sugar. However, the large cell phenotype of the gcr1Delta mutant occurs only in the presence of repressing sugars. GCR1 deletion also results in aberrant derepression of numerous glucose repressed loci; glucose-grown gcr1Delta cells actively respire, demonstrating that this global alteration in transcription corresponds to significant changes at the physiological level. These data offer an insight into the coordination of growth and cell division by providing an integrated view of the transcriptomic, phenotypic, and metabolic consequences of GCR1 deletion.

Carbon↗

PubMed2006

Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei.

Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated.

Animals↗

PubMed2001

Induction of the lipocyte phenotype in murine hepatic stellate cells: reorganisation of the actin cytoskeleton.

Hepatic stellate cells (HSCs) are intralobular connective tissue cells presenting myofibroblast or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix, repair, regeneration and fibrosis under the former phenotype, and control retinol metabolism, storage and release under the latter one. Responding to systemic or local demands, they can convert into the required phenotype with deep modifications of their structures. Using immunofluorescence microscopy and Western blots, we investigated the expression and organisation of actin filaments and of two actin-binding proteins, alpha-actinin and tropomyosin, in the cloned GRX cell line representative of murine HSCs. GRX cells expressing the myofibroblast phenotype showed typical well-organised actin stress-fibres, anchored at the focal adhesions located at the cell periphery. Retinol treatment induced active reorganisation of the cytoskeleton. The major stress fibres were reduced in length, and frequently formed a polygonal meshwork. Subsequently, they fragmented and generated diffuse or granular actin in the perinuclear area, a thin continuous layer around lipid droplets and, in fully converted lipocytes, a peripheral layer of thin actin fibres. alpha-Actinin and tropomyosin were present only in lipocytes, co-distributed with actin in a granular form. Since the cytoskeleton reorganisation preceded lipid accumulation, we conclude that the induction of the lipocyte phenotype represents a full reprogramming of cell gene expression and function. We consider that both the lipocyte and the myofibroblast phenotypes should be considered "activated states" of HSCs, each responding to specific physiological or pathological modifications of liver functions.

Actinin↗