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PubMed2026

Lactylome Reprogramming Mediates Therapeutic Response and Adaptation to Neoadjuvant Chemotherapy in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) exhibits high prevalence in China and poor prognosis despite neoadjuvant chemotherapy (NACT), with significant chemoresistance development. Tumor-associated metabolic reprogramming and NACT-induced cellular stress promote lactate accumulation, which serves as a precursor for lysine lactylation (Kla), a post-translational modification potentially regulating cancer progression. We hypothesized that systematic characterization of the lactylome in response to NACT could reveal critical molecular mechanisms underlying treatment and identify new therapeutic vulnerabilities in ESCC. Herein, through comprehensive proteomic and lactylome profiling of tumor and adjacent normal adjacent tissues from 31 ESCC patients (with or without NACT treatment), we identified 8281 proteins and 1836 Kla sites across 62 samples. NACT induced substantial lactylome alterations with 307 differentially expressed Kla sites predominantly in nonhistone proteins involved in DNA damage response and metabolic pathways. Our data revealed that while NACT-induced suppression of energy metabolism, coupled with upregulated 3-hydroxy-3-methylglutaryl reductase degradation 1 complex expression, may exert potential proapoptotic effects, the activation of ribosome biogenesis and increased nucleoprotein lactylation triggered tumor-protective mechanisms. Mechanistically, we demonstrated that DNA damage and elevated lactate levels induced poly(ADP-ribose) polymerase 1 K654 lactylation, enhancing its enzymatic activity and augmenting poly(ADP-ribosyl)ation of downstream targets, potentially playing a pivotal role in chemotherapy resistance-associated pathways. This comprehensive tissue-level landscape of Kla dynamics in ESCC response to chemotherapy establishes Kla as a critical regulatory mechanism in treatment response, potentially offering novel therapeutic targets and predictive biomarkers for personalized treatment strategies.

Humans↗

PubMed2025

Proteomics and Phosphoproteomics Characteristics of the Rhesus Macaque Lung Infected With Original SARS-CoV-2, Delta, and Omicron Variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains mutate rapidly, making it crucial to study their molecular mechanisms for swift vaccine and drug development. Here, we utilized host lung proteomic and phosphoproteomic profiling to investigate the underlying pathology caused by the variants. Lung tissues infected with wild-type GD108, Delta, or Omicron BA.1 variants showed overexpression of proteins and phosphoproteins linked to the innate immune pathway, particularly in the Omicron group, with high activation of NOD-receptor and RIG-I like receptor signaling pathways. Protein-protein interaction (PPI) analysis revealed six key proteins, including antiviral innate immune response receptor RIG-I (DDX58), and five interferon-related proteins (IFIT2, ISG15, MX1, STAT1, and EIF2AK2), highlighting the importance of the innate immune response in combating all three variants. Kinase prediction analysis suggested that six kinases (DAPK1, DAPK2, DAPK3, PRACK, TTK, and MAP2K2), potentially inhibited by Fostamatinib, were activated across all three variants, and might be potential drug targets, pending further verification. Omicron infection, compared to other mutants, significantly disrupted proteins related to pulmonary structural support, like integrin and collagens, and inhibited efferocytosis, reducing the host's ability to eliminate the pathogen. These findings suggest that innate immune activation and structural disruption may contribute to Omicron-related pathology, potentially being useful for research into the molecular mechanisms underlying lung injury from SARS-CoV-2 variants.

Animals↗

PubMed2026

COVID-19 multi-omics reveal organ-specific responses and biomarkers.

OBJECTIVE: Post-COVID-19 syndrome is characterised by persistent immune dysfunction and multi-organ sequelae. This study aimed to characterise the systemic blood molecular landscape induced by SARS-CoV-2 infection and identify prognostic markers linked to skeletal muscle mass loss, a key driver of poor outcomes. METHODS: We enrolled 30 healthy controls and 307 COVID-19 patients, collecting 422 plasma samples for integrated proteomic and metabolomic profiling to investigate organ-specific molecular alterations in COVID-19. RESULTS: We comprehensively mapped the molecular landscape of COVID-19, encompassing immune, tissue-specific, and metabolic perturbations, and delineated their interactions. Focusing on organ-damage-related molecular patterns associated with disease progression and mortality, we found that skeletal muscle mass loss contributed to poor clinical outcomes of COVID-19 (p < 0.0001). Dysregulated arginine metabolism emerged as a key metabolic signature in fatal COVID-19 cases, with GLUL, GOT1, and citrulline showing significant correlation with skeletal muscle mass loss. Longitudinal analyses further revealed that reduced citrulline levels underlie the poor outcome of COVID-19 patients with muscle mass loss. These findings were robustly supported through multiple approaches: Mendelian randomization confirmed causal relationships between citrulline depletion, sarcopenia/fat-free mass loss, and COVID-19 mortality (p < 0.05), transcriptomic analyses of SARS-CoV-2-infected golden hamsters (GSE231910) provided additional support in enrichment of arginine biosynthesis (FDR < 0.05), and in vitro experiments further demonstrated that citrulline depletion promotes pro-inflammatory M1 macrophage polarisation — a key immunological feature of critical COVID-19. Leveraging these insights, we developed a skeletal muscle loss-specific prognostic prediction model for COVID-19 using GLUL, GOT1, and citrulline. This model effectively stratified patients into high- and low-risk groups (p = 0.035). CONCLUSION: Our study advances the understanding of COVID-19-induced organ pathophysiology and provides a foundation for developing targeted therapeutic strategies for post-COVID sequelae.

COVID-19↗

PubMed2001

Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics.

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.

Amino Acid Sequence↗

PubMed2006

Effects of body weight and nutrition on mammary protein expression profiles in Holstein heifers.

A proteomics approach was used to characterize biochemical and cellular mechanisms governing effects of peripubertal feeding on heifer mammary development. Mammary parenchymal tissue from 24 Holstein heifers randomly assigned to treatments arranged in a 2 x 2 factorial design was used to generate 2-dimensional protein maps of mammary tissue extracts. Heifers were reared on 1 of 2 dietary treatments, restricted (650 g/ d of daily gain) or elevated (950 g/d of daily gain) and killed at 1 of 2 body weights (BW, 200 or 350 kg). Cytosolic mammary gland extracts were prepared from frozen mammary parenchyma. Proteome maps of extracts were constructed using PDQuest software. Densities of 820 protein spots were analyzed using the MIXED procedure of SAS. Protein spots were characterized by changes in profiles of expression in response to increased BW, dietary treatment, or both. Dietary treatment influenced the expression of 131 protein spots, whereas heifer BW influenced the expression of 108 spots. The 22 most highly influenced (statistically) spots were excised and submitted for mass spectrometric analyses. Returned protein names and accession numbers were used in National Center for Biotechnology Information database searches to obtain information on the identified proteins. For example, one of the proteins that differed by dietary treatment, transferrin, a binding protein of insulin-like growth factor binding protein-3, was identified via these methods. Possible roles of this and other proteins in mammary development are described. We concluded that a proteomic approach is an effective tool for identifying the proteins involved in bovine mammary development.

Animal Nutritional Physiological Phenomena↗

PubMed2002

Large-scale mutagenesis and functional genomics in yeast.

One of the major goals for the post-genome era is determining of the function of proteins predicted in the genome sequence. In many organisms functional assignments have been the results of comparative sequencing, proteomics or expression profiling. In the yeast, Saccharomyces cerevisiae, however, the functional role of a gene can be tested directly by disrupting the gene and examining the phenotype of the mutant. Because precise targeted deletions can be easily constructed, it is also possible to systematically delete every gene in the genome. Here we describe recent progress in yeast genome-wide mutagenesis programs and the results produced from analyzing the mutants created by them.

Genes, Lethal↗

PubMed2026

CRISPR-Enabled functional genomics in hPSCs-derived neural models for autism spectrum disorder.

Autism Spectrum Disorder (ASD) is a genetically heterogeneous neurodevelopmental condition in which hundreds of individually rare risk variants converge on a small number of shared biological pathways, including synaptic scaffolding, chromatin remodeling, excitation-inhibition balance, and cellular energy metabolism. Translating this genetic heterogeneity into mechanistic insight requires experimental systems capable of interrogating individual gene functions in human-relevant neural contexts at scale. CRISPR-enabled functional genomics in human pluripotent stem cell (hPSC)-derived neural models, spanning neural progenitors, cortical and inhibitory neurons, astrocytes, microglia, and brain organoids, provides precisely this capability. By integrating pooled perturbation screens with multimodal readouts including single-cell and spatial transcriptomics, chromatin accessibility profiling, proximity labeling proteomics, multi-electrode array electrophysiology, and metabolic flux analysis, these platforms enable systematic, causal mapping of ASD gene function at system resolution. Early applications have already revealed convergent mechanisms: BAF complex disruption expands the ventral progenitor pool and biases its fate toward oligodendrocyte and interneuron lineages; ADNP loss impairs microglial synaptic pruning through altered endocytic trafficking; and mTOR pathway dysregulation in PTEN- and TSC2-perturbed models links genetic risk directly to metabolic and mitochondrial dysfunction. Computational frameworks including MIMOSCA and SCEPTRE enable causal network reconstruction and pseudotime inference from these datasets, moving the field from gene lists toward pathway-level models of ASD pathobiology. Translational applications leverage isogenic iPSC panels and variant-level base and prime editing to stratify ASD variants by functional impact, informing gene therapy design for haploinsufficient targets such as CHD8 and SCN2A via AAV or antisense oligonucleotide delivery. Remaining challenges, including model developmental immaturity, batch variability, and the difficulty of modeling polygenic risk, are addressed by a roadmap integrating spatial perturbomics, AI-driven causal inference, and population-scale standardized biobanks. This review synthesizes the current state of CRISPR-based functional genomics in human stem cell neural models as a coherent experimental framework for converting ASD genetic associations into mechanistic understanding and therapeutic opportunity.

Humans↗

PubMed2004

The role of ductal lavage in the management of women at high risk for breast carcinoma.

Ductal lavage (DL) is a recently introduced technique for nonsurgical breast epithelial sampling of asymptomatic high-risk women. It is based on exfoliative cytology of duct epithelial cells, obtained by cannulation and lavage of ducts that produce nipple fluid after breast massage and nipple aspiration. DL provides information similar to that obtained by cytologic examination of nipple aspiration fluid and random periareolar fine needle aspiration. Women who demonstrate cytologic atypia on these tests can be assumed to be at higher risk for breast cancer and may benefit from prophylactic medication. DL promises to provide more reproducible sampling of the same area of the breast, as opposed to methods of random sampling, such as random fine needle aspiration, but the degree of reproducibility remains to be demonstrated. If reproducibility is greater than with random periareolar fine needle aspiration, DL may represent an important advance in the design of phase II chemoprevention trials, particularly because it also provides another source of material for judging response to prevention agents (ie, the protein component of nipple aspiration fluid), which can be analyzed for levels of hormones, specific proteins, and protein profiles obtained with proteomics. Early data do not suggest that DL is an effective screening tool for breast cancer on the basis of cytologic interpretation of DL samples, although this may change if effective molecular markers are validated for cancer detection in women at high risk.

Biomarkers, Tumor↗

PubMed2026

Integrated analysis reveals the impact of obesity on triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous breast cancer subtype with limited therapeutic options. While the prevalence of overweight/obese (OW/OB) women continues to rise, the impact of obesity on molecular features of TNBC remains incompletely understood. We investigated clinicopathological and molecular data (including genomic, transcriptomic, proteomic and metabolomic profiling) using our original multi-omics database of TNBC (N = 465) for associations with patient body mass index (BMI). Multi-omics profiling revealed that OW/OB patients exhibited worse survival as well as elevated inflammation of tumor microenvironment, higher expression of immune checkpoints, and dysregulated lipid metabolism. Our in vivo experiments demonstrated that tumors in obese mice displayed faster growth rates, a higher proportion of PD-1+CD8+ T cells and enhanced responsiveness to anti-PD-1 treatment. In addition, we analyzed data from four independent clinical trials and discovered that OW/OB patients demonstrated higher pathological complete response rates and longer progression-free survival following anti-PD-1-based immunotherapy. In conclusion, our study systematically revealed that obesity is associated with coordinated immune-metabolic remodeling in TNBC, characterized by checkpoint enrichment and lipid dysregulation, which may help explain the enhanced anti-PD-1 responsiveness and should be taken into account in the field of precision medicine.

Immunity↗

PubMed2004

Adaptation of enzymes to temperature: searching for basic "strategies".

The pervasive influence of temperature on biological systems necessitates a suite of temperature--compensatory adaptations that span all levels of biological organization--from behavior to fine-scale molecular structure. Beginning about 50 years ago, physiological studies conducted with whole organisms or isolated tissues, by such pioneers of comparative thermal physiology as V.Ya. Alexandrov, T.H. Bullock, F.E.J. Fry, H. Precht, C.L. Prosser, and P.F. Scholander, began to document in detail the abilities of ectothermic animals to sustain relatively similar rates of metabolic activity at widely different temperatures of adaptation or acclimation. These studies naturally led to investigation of the roles played by enzymatic proteins in metabolic temperature compensation. Peter Hochachka's laboratory became an epicenter of this new focus in comparative physiology. The studies of the enzyme lactate dehydrogenase (LDH) that he initiated as a PhD student at Duke University in the mid-1960s and continued for several years at the University of British Columbia laid much of the foundation for subsequent studies of protein adaptation to temperature. Studies of orthologs of LDH have revealed the importance of conserving kinetic properties (catalytic rate constants (kcat) and Michaelis-Menten constants (Km) and structural stability during adaptation to temperature, and recently have identified the types of amino acid substitutions causing this adaptive variation. The roles of pH and low-molecular-mass organic solutes (osmolytes) in conserving the functional and structural properties of enzymes also have been elucidated using LDH. These studies, begun in Peter Hochachka's laboratory almost 40 years ago, have been instrumental in the development of a conceptual framework for the study of biochemical adaptation, a field whose origin can be traced largely to his creative influences. This framework emphasizes the complementary roles of three "strategies" of adaptation: (1) changes in amino acid sequence that cause adaptive variation in the kinetic properties and stabilities of proteins, (2) shifts in concentrations of proteins, which are mediated through changes in gene expression and protein turnover; and (3) changes in the milieu in which proteins function, which conserve the intrinsic properties of proteins established by their primary structure and modulate protein activity in response to physiological needs. This theoretical framework has helped guide research in adaptational biochemistry for many years and now stands poised to play a critical role in the post-genomic era, as physiologists grapple with the challenge of integrating the wealth of new data on gene sequences (genome), gene expression (transcriptome and proteome), and metabolic profiles (metabolome) into a realistic physiological context that takes into account the evolutionary histories and environmental relationships of species.

Adaptation, Physiological↗

PubMed2026

Ketogenic diet dampens excitatory neurotransmission by shrinking synaptic vesicle pools.

Ketogenic diet (KD) is used for the treatment of drug-resistant childhood epilepsy and has been proposed to improve outcomes in neurodegenerative diseases. However, the mechanisms by which KD alters brain circuitry remain unclear. Here, we investigated the impact of KD on hippocampal function through integrative analysis of gene expression and neurotransmission. We found that KD induces extensive transcriptional reprogramming, including altered expression of numerous synaptic genes. Proteomic and genomic profiling revealed significant changes in histone modifications, particularly at promoters of KD-regulated genes. Electrophysiological recordings showed that KD reduces excitatory synaptic gain and short-term plasticity at CA3-CA1 synapses, dampening the summation of excitatory inputs and enhancing the summation of inhibitory inputs. These functional changes were driven, in part, by a reduction in the readily releasable vesicle pool at excitatory synapses under KD. Together, our findings demonstrate that KD drives transcriptional remodeling of hippocampal circuits, leading to synaptic adaptations that may underlie its anti-epileptic and neuroprotective effects.

Animals↗

PubMed2005

Micro-separation toward systems biology.

Current biology is experiencing transformation in logic or philosophy that forces us to reevaluate the concept of cell, tissue or entire organism as a collection of individual components. Systems biology that aims at understanding biological system at the systems level is an emerging research area, which involves interdisciplinary collaborations of life sciences, computational and mathematical sciences, systems engineering, and analytical technology, etc. For analytical chemistry, developing innovative methods to meet the requirement of systems biology represents new challenges as also opportunities and responsibility. In this review, systems biology-oriented micro-separation technologies are introduced for comprehensive profiling of genome, proteome and metabolome, characterization of biomolecules interaction and single cell analysis such as capillary electrophoresis, ultra-thin layer gel electrophoresis, micro-column liquid chromatography, and their multidimensional combinations, parallel integrations, microfabricated formats, and nano technology involvement. Future challenges and directions are also suggested.

Chromatography, Liquid↗

PubMed2026

A distinct effector B cell population drives autoantibody production in SARS-CoV-2 infection.

Autoantibodies (autoAbs) are linked to mortality and Long COVID, yet their cellular origins remain unclear. We analyzed the INCOV cohort and identified 12 age- and sex-matched participants with varying autoAb abundance and integrated single-cell RNA-seq and ATAC-seq data from B cells, plasma proteomics, proteome-wide autoAb profiling, clinical data, and in vitro assays. AutoAb abundance inversely correlated with neutralizing IgG and declined as infection resolved, paralleling the contraction of atypical memory B cells (AtMs). In vitro, AtMs preferentially differentiated into autoAb-producing antibody-secreting cells upon TLR7/8 stimulation. CD11c+ AtMs (double-negative 2, DN2s) in autoAb-high individuals exhibited increased TLR7 signaling, oxidative stress, and isotype switching, regulated by transcription factors T-bet and XBP1. Integrated genetic and genomic analyses showed that DN2s had the strongest enrichment for autoimmune trait heritability and inferred regulatory effects of autoimmune risk variants among B cell subsets. These findings identify DN2s as key precursors of autoAb-producing cells during SARS-CoV-2 infection.

B cell↗

PubMed2002

Functional screening of intracellular proteins in single cells and in patterned cell arrays using electroporation.

A tool for detection and characterization of intracellular enzyme-substrate and receptor-ligand interactions inside the cytoplasm of single targeted cells or small confined groups of cells is presented. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered by electroosmosis and internalized by electroporation in cells using an electrolyte-filled capillary (EFC) biased at a high voltage. Specifically, alkaline phosphatase and proteases were detected in single NG108-15 cells using fluorescein diphosphate and casein BODIPY FL, respectively. The intracellular 1,4,5-inositol triphosphate (IP3) and ryanodine receptors were detected after EFC introduction of the selective receptor agonists IP3 and cyclic adenosine diphosphate ribose (cADPr), respectively. Receptor activation in both cases resulted in increased cytosolic concentrations of free calcium ions that were measured using the calcium-ion-selective probe, fluo-3. The effect of cADPr could be blocked by coadministration of the ryanodine receptor antagonist ruthenium red. Furthermore, electroporation of a plurality of cells grown in microwell structures (100 x 100 x 45 microm) molded in PDMS is demonstrated. The methods and systems described using an EFC for electroporation and delivery of protein markers, ligands, and substrates might be useful in high-throughput screening of intracellular targets, with applications in proteomics and phenotype profiling, as well as in drug discovery.

Alkaline Phosphatase↗

PubMed2002

Mechanisms in epithelial plasticity and metastasis: insights from 3D cultures and expression profiling.

Most human tumors are of epithelial origin (carcinomas) and metastases from such tumors lead to >80% of all cancer deaths. In contrast to aberrant control of proliferation, cell cycle, apoptosis, angiogenesis, and lifespan, mechanisms involved in local invasion and metastasis are still insufficiently understood. We will review a set of (often conflicting) in vitro/in vivo data that suggest the existence of several types of epithelial cell plasticity changes towards a fibroblastoid, invasive phenotype, which increasingly emerge as crucial events during metastasis. New cellular models were identified, which form organotypic structures under near-physiological 3D-culture conditions in vitro as well as tumors/metastases in vivo. In these models, key proteins and signaling pathways were identified (e.g., TGFbeta, ERK/MAPK, PI3K, and PDGF), which specify distinct types of epithelial plasticity correlated with steps in cancer progression and metastasis. The existence of several distinct epithelial plasticity phenotypes is also strongly suggested by expression profiling of polysome-bound mRNA, yielding a better representation of the proteome than conventional expression profiling.

Animals↗

PubMed2026

Scalable, generalizable and uncertainty-aware integration of spatial multiomics across diverse modalities and platforms with SCIGMA.

Recent advances in spatial omics technologies have enabled simultaneous profiling of transcriptomic, proteomic, epigenomic, metabolomic and imaging data at high spatial resolution, offering unprecedented opportunities to dissect tissue complexity. However, integrating these diverse and large-scale spatial multimodal datasets remains a major computational challenge. We present SCIGMA, a scalable and generalizable deep learning framework for spatial multiomics integration. SCIGMA introduces an uncertainty-aware contrastive learning objective and multiview graph neural networks to preserve modality-specific signals while learning biologically meaningful joint representations. Unlike previous methods, SCIGMA provides spatially resolved uncertainty estimates, interpretably identifying regions of biological or technical heterogeneity. SCIGMA supports integration of up to five modalities, and its modular framework is extensible to future technologies with even more modalities. It also scales to more than 1 million spatial locations, enabling analysis of high-resolution datasets such as Visium HD and Xenium Prime. We evaluated SCIGMA across 19 datasets spanning 8 modalities, 10 tissues and 9 platforms. On benchmarkable datasets, SCIGMA outperformed other methods in spatial domain detection, modality preservation, feature reconstruction and reproducibility. SCIGMA identifies biologically meaningful structures, refined spatial domains and modality-specific regulatory programs, providing a robust, flexible and future-ready solution for scalable spatial multimodal integration.

Multiomics↗

PubMed2025

DNA methylation at retrotransposons protects the germline by preventing NRF1-mediated activation.

Silencing evolutionary young retrotransposons by cytosine DNA methylation is essential for spermatogenesis, as failure to methylate their promoters leads to reactivation, meiotic failure, and infertility. How retrotransposons reactivate in the absence of DNA methylation is poorly understood. We show that upon defective DNA methylation, distinct retrotransposon families display unique expression patterns and chromatin landscapes during mouse spermatogenesis. We find that their reactivation in meiotic spermatocytes correlates with the loss of bivalent H3K4me3-H3K27me3 chromatin marks. Through proteomics and chromatin profiling, we identify NRF1 as a DNA methylation-sensitive transcription factor that transactivates unmethylated retrotransposons. Conditional germline knockout of Nrf1 in the absence of DNA methylation rescues the silencing of the most mutagenic retrotransposon in mice, namely Intracisternal A-particle or IAP. Our findings reveal that chromatin modifications together with a DNA methylation-sensitive transcription factor regulate retrotransposon expression in the absence of DNA methylation in spermatogenesis, revealing a mechanism by which retrotransposons proliferate in the germline after evading DNA methylation-based silencing.

Animals↗

PubMed2004

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed.

Amino Acid Sequence↗