Biochemical Signal Transduction Pathways Research Articles

Evaluation of the Immune Response of Patulin by Proteomics.

Patulin, an emerging mycotoxin with high toxicity, poses great risks to public health. Considering the poor antibody production in patulin immunization, this study focuses on the four-dimensional data-independent acquisition (4D-DIA) quantitative proteomics to reveal the immune response of patulin in rabbits. The rabbit immunization was performed with the complete developed antigens of patulin, followed by the identification of the immune serum. A total of 554 differential proteins, including 292 up-regulated proteins and 262 down-regulated proteins, were screened; the differential proteins were annotated; and functional enrichment analysis was performed. The differential proteins were associated with the pathways of metabolism, gene information processing, environmental information processing, cellular processes, and organismal systems. The functional enrichment analysis indicated that the immunization procedures mostly resulted in the regulation of biochemical metabolic and signal transduction pathways, including the biosynthesis of amino acid (glycine, serine, and threonine), ascorbate, and aldarate metabolism; fatty acid degradation; and antigen processing and presentation. The 14 key proteins with high connectivity included G1U9T1, B6V9S9, G1SCN8, G1TMS5, G1U9U0, A0A0G2JH20, G1SR03, A0A5F9DAT4, G1SSA2, G1SZ14, G1T670, P30947, P29694, and A0A5F9C804, which were obtained by the analysis of protein-protein interaction networks. This study could provide potential directions for protein interaction and antibody production for food hazards in animal immunization.

RNA Sequencing and Related Differential Gene Expression Analysis in a Mouse Model of Emphysema Induced by Tobacco Smoke Combined with Elastin Peptides.

To establish a model of emphysema induced by tobacco smoke combined with elastin peptides (EP), explore the biochemical metabolic processes and signal transduction pathways related to emphysema occurrence and development at the transcriptional level, and identify new targets and signaling pathways for emphysema prevention and treatment. Mice were randomly divided into the air pseudoexposure group (NORMAL group) and the tobacco smoke + EP group (EP group). The differentially expressed genes (DEGs) in lung tissue between the two groups were identified by RNA-seq, and functional annotation and Gene Ontology (GO)/ Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. The differential expression of the selected genes were verified using qRT‒PCR and immunohistochemistry (IHC). EP group mice showed emphysema-like changes. The expression levels of 1159 genes in the EP group differed significantly (529 up-regulated and 630 down-regulated) from those in the NORMAL group. GO enrichment analysis showed that the DEGs were significantly enriched in the terms immune system, adaptive immune response, and phosphorylation, while KEGG pathway enrichment analysis showed that the DEGs were enriched mainly in the pathways cytokine‒cytokine receptor interaction, T-cell receptor signaling pathway, MAPK signaling pathway, Rap1 signaling pathway, endocytosis, chemokine signaling pathway, Th17 cell differentiation, and Th1 and Th2 cell differentiation. The differential expression of the selected DEGs were verified by qRT‒PCR and IHC, and the expression trends of these genes were consistent with those identified by RNA-seq. Emphysema may be related to the inflammatory response, immune response, immune regulation, oxidative stress injury, and other biological processes. The Bmp4-Smad-Hoxa5/Acvr2a signaling pathway may be involved in COPD/ emphysema occurrence and development.

Information transfer in mammalian glycan-based communication.

Glycan-binding proteins, so-called lectins, are exposed on mammalian cell surfaces and decipher the information encoded within glycans translating it into biochemical signal transduction pathways in the cell. These glycan-lectin communication pathways are complex and difficult to analyze. However, quantitative data with single-cell resolution provide means to disentangle the associated signaling cascades. We chose C-type lectin receptors (CTLs) expressed on immune cells as a model system to study their capacity to transmit information encoded in glycans of incoming particles. In particular, we used nuclear factor kappa-B-reporter cell lines expressing DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), macrophage C-type lectin (MCL), dectin-1, dectin-2, and macrophage-inducible C-type lectin (MINCLE), as well as TNFαR and TLR-1&2 in monocytic cell lines and compared their transmission of glycan-encoded information. All receptors transmit information with similar signaling capacity, except dectin-2. This lectin was identified to be less efficient in information transmission compared to the other CTLs, and even when the sensitivity of the dectin-2 pathway was enhanced by overexpression of its co-receptor FcRγ, its transmitted information was not. Next, we expanded our investigation toward the integration of multiple signal transduction pathways including synergistic lectins, which is crucial during pathogen recognition. We show how the signaling capacity of lectin receptors using a similar signal transduction pathway (dectin-1 and dectin-2) is being integrated by compromising between the lectins. In contrast, co-expression of MCL synergistically enhanced the dectin-2 signaling capacity, particularly at low-glycan stimulant concentration. By using dectin-2 and other lectins as examples, we demonstrate how signaling capacity of dectin-2 is modulated in the presence of other lectins, and therefore, the findings provide insight into how immune cells translate glycan information using multivalent interactions.

Differential expression of microRNAs in bile duct obstruction-induced liver fibrosis and the identification of a novel liver fibrosis marker miR-1295b-3p.

Bile duct obstruction-induced liver fibrosis is mainly caused by cholestatic liver injury which stimulates liver cell inflammation and damages the liver structure, causing liver fibrosis. The differentially expressed microRNAs and the potential target genes and signal pathways that are involved in bile duct obstruction-induced liver fibrosis remain unclear. We examined the differential expression of microRNAs and the target genes in the liver tissues of patients with liver fibrosis. High-throughput sequencing was used to detect the total microRNAs and identify the differentially expressed microRNAs. The topGO software was used to perform the Gene Ontology (GO) function enrichment analysis. The KOBAS software was used to analyze the associated biochemical metabolic pathways and signal transduction pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were conducted to detect the expression of miR-1295b-3p, alpha smooth muscle actin (α-SMA), Bcl-2, caspase-3, Bax, and β-arrestin1 (ARRB1). Cell viability and apoptosis were detected by the Cell Counting Kit 8 (CCK-8) assay and flow cytometry. The targeting relationship between ARRB1 and miR-1295b-3p was verified using luciferase reporter assays. A total of 44 microRNAs were found to be differentially expressed, including 18 upregulated and 26 downregulated microRNAs. Five downregulated microRNAs, including miR-483-3p, miR-5589-3p, miR-1271-5p, miR-1295b-3p, and miR-7977. GO functional enrichment analysis of the target genes revealed the molecular functions, cellular location, and biological processes involved. Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis showed that the target genes are mainly involved in metabolic pathways. In addition, the results of qRT-PCR revealed that miR-1295b-3p was downregulated in human fibrotic liver tissues and TGF-β1-activated LX-2 cells (human hepatic stellate cell line). Overexpression of miR-1295b-3p alleviated liver fibrosis, decreased the α-SMA levels, and inhibited proliferation and enhanced apoptosis in LX-2 cells. Dual-luciferase assays revealed that miR-1295b-3p suppressed ARRB1 expression by binding directly to its 3' untranslated region (UTR). This study identified the differentially expressed microRNAs in bile duct obstruction-induced liver fibrosis and revealed the potential target genes and signal pathways involved. Overexpression of miR-1295b-3p alleviated liver fibrosis, however, the specific targeting mechanisms warrant further clarification. Therefore, overexpressing miR-1295b-3p may be a potential treatment method for liver fibrosis.

Application value of plasma proteomics in the diagnosis and pathogenesis of high altitude polycythemia

Objective: To explore the application value of plasma proteomics in the diagnosis and pathogenesis of high altitude polycythemia (HAPC). Methods: Ten patients with HAPC in Qinghai Provincial People's Hospital from January 2020 to January 2021 were selected as the experimental group, including 4 males and 6 females, aged (46±4) years. Ten healthy controls at the same altitude in the same period were selected as the control group, including 5 males and 5 females, aged (44±4) years. The differential proteins were identified and quantified by high performance liquid chromatography-mass spectrometry (HPLC-MS), and the gene ontology (GO) functional enrichment analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis and interaction network analysis were conducted for the selected differential proteins. Results: A total of 117 differential proteins with quantitative values were screened from the experimental group and the control group, 45 significantly up-regulated proteins with quantitative values only in the experimental group, 40 significantly down-regulated proteins with quantitative values only in the control group, and 32 differentially expressed proteins with quantitative values in both experimental and control groups were detected. Compared with the control group, 11 of the 32 differentially expressed proteins in the experimental group were down-regulated and 21 were up-regulated. The results of GO functional enrichment analysis showed that the biological processes involved by differential proteins mainly included immune response, complement activation, activated protein cascade and coagulation system. The results of KEGG function enrichment analysis showed that the main biochemical metabolic pathways and signal transduction pathways involved by differential proteins were axon guidance, lysosomes, cell adhesion molecules, lipid and atherosclerosis, hematopoietic cell lineage and cholesterol metabolism. The abundant domains are mainly in immunoglobulin-like domain, EGF-like domain, fibronectin type Ⅲ superfamily, serine proteases, Sushi/SCR/CCP superfamily. The results of differential protein interaction analysis showed that the interaction score was>700, and the top 10 differential proteins with the largest number of nodes were MPO, RPS27A, ARG1, GM2A, TIMP1, CRP, FABP5, HBB, S100A7 and RHOA, respectively. Conclusion: Plasma proteomics analysis technique is helpful to identify the related protein markers in the development of HAPC, and provide reference for the diagnosis and pathogenesis of HAPC.

Geographical Origin Differentiation of Rice by LC-MS-Based Non-Targeted Metabolomics.

Many factors, such as soil, climate, and water source in the planting area, can affect rice taste and quality. Adulterated rice is common in the market, which seriously damages the production and sales of high-quality rice. Traceability analysis of rice has become one of the important research fields of food safety management. In this study, LC-MS-based non-targeted metabolomics technology was used to trace four rice samples from Heilongjiang and Jiangsu Provinces, namely, Daohuaxiang (DH), Huaidao No. 5 (HD), Songjing (SJ), and Changlixiang (CL). Results showed that the discrimination accuracy of the partial least squares discriminant analysis (PLS-DA) model was as high as 100% with satisfactory prediction ability. A total of 328 differential metabolites were screened, indicating significant differences in rice metabolites from different origins. Pathway enrichment analysis was carried out on the four rice samples based on the KEGG database to determine the three metabolic pathways with the highest enrichment degree. The main biochemical metabolic pathways and signal transduction pathways involved in differential metabolites in rice were obtained. This study provides theoretical support for the geographical origins of rice and elucidates the change mechanism of rice metabolic pathways, which can shed light on improving rice quality control.

Cyclic Nucleotide (cNMP) Analogues: Past, Present and Future.

Cyclic nucleotides are important second messengers involved in cellular events, and analogues of this type of molecules are promising drug candidates. Some cyclic nucleotide analogues have become standard tools for the investigation of biochemical and physiological signal transduction pathways, such as the Rp-diastereomers of adenosine and guanosine 3′,5′-cyclic monophosphorothioate, which are competitive inhibitors of cAMP- and cGMP-dependent protein kinases. Next generation analogues exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity, or are caged or photoactivatable for fast and/or targeted cellular imaging. Novel specific nucleotide analogues activating or inhibiting cyclic nucleotide-dependent ion channels, EPAC/GEF proteins, and bacterial target molecules have been developed, opening new avenues for basic and applied research. This review provides an overview of the current state of the field, what can be expected in the future and some practical considerations for the use of cyclic nucleotide analogues in biological systems.

Microbiome analysis, the immune response and transplantation in the era of next generation sequencing

The human gastrointestinal tract, skin and mucosal surfaces are inhabited by a complex system of bacteria, viruses, fungi, archaea, protists, and eukaryotic parasites with predominance of bacteria and bacterial viruses (bacteriophages). Collectively these microbes form the microbiota of the microecosystem of humans. Recent advancement in technologies for nucleic acid isolation from various environmental samples, feces and body secretions and advancements in shotgun throughput massive parallel DNA and RNA sequencing along with 16S ribosomal gene sequencing have unraveled the identity of otherwise unknown microbial entities constituting the human microecosystem. The improved transcriptome analysis, technological developments in biochemical analytical methods and availability of complex bioinformatics tools have allowed us to begin to understand the metabolome of the microbiome and the biochemical pathways and potential signal transduction pathways in human cells in response to microbial infections and their products. Also, developments in human whole genome sequencing, targeted gene sequencing of histocompatibility genes and other immune response associated genes by Next Generation Sequencing (NGS) have allowed us to have a better conceptualization of immune responses, and alloimmune responses. These modern technologies have enabled us to dive into the intricate relationship between commensal symbiotic and pathogenic microbiome and immune system. For the most part, the commensal symbiotic microbiota helps to maintain normal immune homeostasis besides providing healthy nutrients, facilitating digestion, and protecting the skin, mucosal and intestinal barriers. However, changes in diets, administration of therapeutic agents like antibiotics, chemotherapeutic agents, immunosuppressants etc. along with certain host factors including human histocompatibility antigens may alter the microbial ecosystem balance by causing changes in microbial constituents, hierarchy of microbial species and even dysbiosis. Such alterations may cause immune dysregulation, breach of barrier protection and lead to immunopathogenesis rather than immune homeostasis. The effects of human microbiome on immunity, health and disease are currently under intense research with cutting edge technologies in molecular biology, biochemistry, and bioinformatics along with tremendous ability to characterize immune response at single cell level. This review will discuss the contemporary status on human microbiome immune system interactions and their potential effects on health, immune homeostasis and allograft transplantation.

The Role of TGFβ in Clinical Cancer Response

The role of transforming growth factor beta (TGFβ) is context dependent. Even within the specific context of cancer, the functional significance of TGFβ varies according to environmental conditions. Therapies targeting TGFβ capitalize upon recent discoveries in canonical SMAD-dependent biochemical signaling networks as well as non-canonical SMAD-independent biochemical signal transduction pathways and mechanobiological signal transduction pathways. TGFβ clinical trials utilizing vaccine, small molecule inhibitor, antibody, and aptamer strategies support TGFβ as a viable anticancer target for a variety of cancer diagnoses. Combination therapies incorporating TGFβ inhibition and immune checkpoint blockade provide additional avenues for future TGFβ-based clinical intervention.

STEM-14CELLULAR RESTING TRANSMEMBRANE POTENTIAL INSTRUCTS DIFFERENTIATION STATE OF GBM STEM-LIKE CELLS

INTRODUCTION: Glioblastoma (GBM) is the most common malignant brain tumor in adults with a poor prognosis despite maximal treatment. Resting membrane potential (RMP) has been shown to be an important regulator of the differentiation state of embryonic and somatic stem cells. We evaluated the relevance of RMP in GBM stem-like cells (GSC). METHODS: Whole cell patch clamp technique was used to determine the RMP. RESULTS: Undifferentiated GSC had a RMP of -5mV +/-4 (n = 33) compared to their differentiated counterparts, which had a mean RMP of -24mV +/-20 (n = 31, P < 0.001). To determine whether the cellular RMP played an instructive role in the differentiation state of GSC we used pharmacologic and genetic approaches to alter the potential. We added additional potassium chloride (KCl) to differentiated GCS in order to induce depolarization, and compared to osmotic controls using sodium chloride (NaCl). We found that GSC exposed to high KCl retained stem markers and sphere forming capacity compared to controls. We then examined whether hyperpolarization was sufficient to induce differentiation. We treated GSC with vacuolar ATPase inhibitors and salinomycin (potassium ionophore), which induced relative hyperpolarization of GSC and induction of differentiation. Transduction of GSC with inducible potassium ion channel led to hyperpolarization and differentiation. To identify potential mechanisms coupling electrical alterations to biochemical signal transduction pathways we performed extensive phospho-protein array studies comparing the undifferentiated GSC and cells hyperpolarized with omeprazole or salinomycin. Attenuation of key developmental and pro-growth signaling pathways was observed. CONCLUSIONS: Alteration of RMP may represent a new therapeutic strategy.

Tuning cell–surface affinity to direct cell specific responses to patterned proteins

Interactions with local extracellular cues direct cell migration. A versatile method to study cell response to a protein consists of patterning the protein cue on a substrate and quantifying the distribution of cells between patterned and non-patterned areas. Here, we define the concepts of (i) cell–surface affinity to describe cell choices, and of (ii) reference surface (RS) to clarify that the choice is made relative to a reference. Furthermore, we report a method to systematically tune the RS and show that it can dominate the experimental cell response to a protein cue. The cell response to a cue can be switched from strong preference to strong aversion by only changing the RS. Using microcontact printing, we patterned the extracellular matrix proteins fibronectin or netrin-1 adjacent to a series of RSs with different ratios of poly-d-lysine (PDL) and polyethylene glycol (PEG), which are of high affinity and of low-affinity for cells, respectively. C2C12 myoblasts or primary neurons seeded on substrates with a high affinity RS (high % PDL) did not respond to a printed protein of interest, and conversely on RSs of low affinity (high % PEG) the cells preferred the printed protein even in the absence of a specific interaction. However, when testing cell response to a standardized series of RSs varying from high to low affinity, a specific response curve was obtained that was unique to each cell type–protein pair. Importantly, for intermediate RSs with moderate affinity, the cell response to the cue was dependent on the activation of biologically relevant protein-specific biochemical signal transduction pathways. Our results establish that choices made by cells in response to a surface-bound cue must take into account, and be interpreted in the context of, the RS. The use of a series of RSs with varying cell–surface affinity reveals specific response curves of cells to a cue that can be compared quantitatively and that may help gain new insights into cellular responses to extracellular proteins.

LIGAMENT ENGINEERING: CHARACTERISTICS OF BETA FIBROBLAST GROWTH FACTOR RELEASE FROM A BIOENGINEERED SCAFFOLD.

The anterior cruciate ligament is a commonly injured ligament of the knee in which ligament reconstruction with a graft is necessary due to its limited healing capacity. With limitations on the existing graft alternatives, we aim to engineer a ligament using a donor9s bone marrow stromal cells (BMSCs). Recently, our laboratory reported synergism between biomechanical and biochemical stimulation (bFGF) resulting in up-regulation of gene expression of stress-resistant collagen I and stress-responsive collagen III and tenascin-C. However, it was unclear if this observation was due to true synergism between mechanical and biochemical signal transduction pathways or due to enhanced release kinetics of the bFGF. The objective of this project is to determine the effects of mechanical stimulation on the release kinetics of bFGF in the presence of a complex biologic environment (cells, media, etc.). Microporous scaffolds were constructed from biodegradable polycaprolactone polymers, loaded with 400 ng I-125-labeled bFGF per scaffold and seeded with BMSCs harvested from rats. Release of bFGF was quantified by measuring radioactivity from I-125-labeled bFGF on either strained (6%) or unstrained scaffolds. After 10 hours, approximately 30% of the bFGF was burst-released from the scaffolds, 10% of bFGF was released after 6 days, and approximately 60% remained on the scaffolds. Mechanical stimulation did not affect the release of bFGF within this complex biologic environment. In summary, we have established and characterized a controlled release system to deliver bFGF to cells seeded within microporous scaffolds and subjected to biomechanical stimulation. Given that the release kinetics are not influenced by mechanical stimulation, this system allows us to uncouple the effects of mechanical stimulation and growth factor delivery in future experiments. This finding also encourages the investigation of common signal transduction pathways that may be involved in the observed synergism between biomechanical and biochemical stimulation.

Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress

Sclerotium-forming filamentous fungi are of great agricultural and biological interest because they can be viewed as models of simple metamorphosis. They differentiate by asexually producing sclerotia but the processes involved in sclerotial metamorphosis were poorly understood. In 1997, it was shown for the first time that the sclerotial differentiation state in Sclerotium rolfsii concurred with increasing levels of lipid peroxides. This finding prompted the development of a theory supporting that sclerotial metamorphosis is induced by oxidative stress. Growth factors that reduce or increase oxidative stress are expected to inhibit or promote sclerotium metamorphosis, respectively. This theory has been verified by a series of published data on the effect of certain hydroxyl radical scavengers on sclerotial metamorphosis, on the identification and quantification of certain endogenous antioxidants (such as ascorbic acid, β-carotene) in relation to the fungal undifferentiated and differentiated states, and on their inhibiting effect on sclerotial metamorphosis as growth nutrients. In 2004-2005, we developed assays for the measurement of certain redox markers of oxidative stress, such as the thiol redox state, the small-sized fragmented DNA, and the superoxide radical. These new advances allowed us to initiate studies on the exact role of glutathione, hydrogen peroxide, and superoxide radical on sclerotial metamorphosis. The emerging data, combined with similar data from other better-studied fungi, allowed us to make some preliminary postulations on the ROS-dependent biochemical signal transduction pathways in sclerotiogenic filamentous fungi.

Urocortin-Induced Inhibition of Muscle Protein Breakdown in Sepsis

Skeletal muscle atrophy is a catabolic condition observed in several pathological conditions including sepsis. Recent studies have shown that activity at the corticotropin releasing factor receptor 2 (CRFR2) can modulate muscle mass. Although, treatment of rats with the CRFR2 agonist, urocortin, inhibited casting-induced skeletal muscle atrophy, the biochemical mechanisms and signal transduction pathways leading to this effect are unknown. We determined whether urocortin treatment would reduce sepsis-induced protein breakdown and investigated the signaling pathways associated with this inhibition of catabolism. Male Sprague-Dawley (SD) rats weighing ∼60g were anesthetized and randomized to caecal ligation and puncture (CLP) or sham-surgery. Sixteen hrs later, we determined protein breakdown in extensor digitorum longus (EDL) muscles by an in vitro tyrosine release method. EDL muscles were isolated from CLP or sham-operated rats, and treated with urocortin, or urocortin + theophylline for 2 hrs prior to freezing in liquid nitrogen. Total RNA was isolated, and mRNA...

HIF-1&amp;#945; and p53 as Targets of NO in Affecting Cell Proliferation, Death and Adaptation

During the past years nitric oxide (NO) signaling became an integral component in understanding physiological and pathophysiological processes of cell proliferation, death or cellular adaptation. Among other activities NO affects multiple targets that allow regulation of gene expression. Although there is no evidence for direct NO-responsive DNA elements within promotor regions of eukaryotic genes numerous indirect signaling pathways exist to explain NO-regulated gene expression. A characteristic feature of some transcription factors such as hypoxia inducible factor-1alpha (HIF-1alpha) or p53 (tumor suppressor p53) is their low protein abundance in unstressed cells due to efficient 26S proteasomal degradation of the protein. Characteristically, the protein amount of HIF-1alpha or p53 is increased steeply upon hypoxic stress or mechanisms that require activation of "guardian of the genome", i.e. p53. Current available data illustrate that NO is endowed with the ability to mimic a hypoxic response by stabilizing HIF-1alpha and/or to accumulate p53 and thus to affect viability decisions. Here we review recent advances in understanding molecular mechanisms how NO affects stability regulation of HIF-1alpha and p53. Moreover, we summarize existing concepts how HIF-1alpha and p53 interact to direct proliferation, death or adaptation. Considering HIF-1alpha and p53 as targets of reactive nitrogen intermediates (RNI) may provide insights into basic chemical reactions, biochemical signal transduction pathways with broad implications for medicine.

The role of nitric oxide (NO) in stability regulation of hypoxia inducible factor-1alpha (HIF-1alpha).

Hypoxia inducible factor-1 (HIF-1) is a master regulator under conditions of decreased oxygen availability. As a hypoxia inducible transcription factor HIF-1 is a heterodimer composed of the helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) proteins HIF-1alpha and the aryl hydrocarbon nuclear translocator (ARNT) also known as HIF-1beta. The HIF-1 transcriptional system senses decreased oxygen availability and transmits this signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, an altered energy metabolism, as well as cell survival decisions. Among recent advances are the discoveries that reactive nitrogen species (RNS), oxygen species (ROS), cytokines, and growth factors participate in stability regulation of HIF-1alpha and HIF-1 transactivation during normoxia. Here we summarize current knowledge and existing concepts that help to understand how NO affects protein accumulation of HIF-1alpha. Considering the fundamental role of radicals, especially NO, as signaling molecules makes HIF-1alpha an attractive target under conditions of NO formation that may be attributed to both, physiology and pathology. Although initial observations showed that NO inhibits hypoxia-induced HIF-1alpha stabilization and HIF-1 transcriptional activation, later studies indicated that exposure of various cells to chemically diverse NO donors or conditions of endogenous NO formation under normoxic conditions induced HIF-1alpha accumulation, HIF-1-DNA binding, and activation of downstream target gene expression. These contrasting situations evoked by NO provide insights into basic chemical reactions, biochemical signal transduction pathways with broad implications for medicine.

Models of Eukaryotic Gradient Sensing: Application to Chemotaxis of Amoebae and Neutrophils

Eukaryotic cells can detect shallow gradients of chemoattractants with exquisite precision and respond quickly to changes in the gradient steepness and direction. Here, we describe a set of models explaining both adaptation to uniform increases in chemoattractant and persistent signaling in response to gradients. We demonstrate that one of these models can be mapped directly onto the biochemical signal-transduction pathways underlying gradient sensing in amoebae and neutrophils. According to this scheme, a locally acting activator (PI3-kinase) and a globally acting inactivator (PTEN or a similar phosphatase) are coordinately controlled by the G-protein activation. This signaling system adapts perfectly to spatially homogeneous changes in the chemoattractant. In chemoattractant gradients, an imbalance between the action of the activator and the inactivator results in a spatially oriented persistent signaling, amplified by a substrate supply-based positive feedback acting through small G-proteins. The amplification is activated only in a continuous presence of the external signal gradient, thus providing the mechanism for sensitivity to gradient alterations. Finally, based on this mapping, we make predictions concerning the dynamics of signaling. We propose that the underlying principles of perfect adaptation and substrate supply-based positive feedback will be found in the sensory systems of other chemotactic cell types.

Neuronal cell death in nervous system development, disease, and injury (Review)

Neuronal death is normal during nervous system development but is abnormal in brain and spinal cord disease and injury. Apoptosis and necrosis are types of cell death. They are generally considered to be distinct forms of cell death. The re-emergence of apoptosis may contribute to the neuronal degeneration in chronic neurodegenerative disease, such as amyotrophic lateral sclerosis and Alzheimer's disease, and in neurological injury such as cerebral ischemia and trauma. There is also mounting evidence supporting an apoptosis-necrosis cell death continuum. In this continuum, neuronal death can result from varying contributions of coexisting apoptotic and necrotic mechanisms; thus, some of the distinctions between apoptosis and necrosis are becoming blurred. Cell culture and animal model systems are revealing the mechanisms of cell death. Necrosis can result from acute oxidative stress. Apoptosis can be induced by cell surface receptor engagement, growth factor withdrawal, and DNA damage. Several families of proteins and specific biochemical signal-transduction pathways regulate cell death. Cell death signaling can involve plasma membrane death receptors, mitochondrial death proteins, proteases, kinases, and transcription factors. Players in the cell death and cell survival orchestra include Fas receptor, Bcl-2 and Bax (and their homologues), cytochrome c, caspases, p53, and extracellular signal-regulated protein kinases. Some forms of cell death require gene activation, RNA synthesis, and protein synthesis, whereas others forms are transcriptionally-translationally-independent and are driven by posttranslational mechanisms such as protein phosphorylation and protein translocation. A better understanding of the molecular mechanisms of neuronal cell death in nervous system development, injury and disease can lead to new therapeutic approaches for the prevention of neurodegeneration and neurological disabilities and will expand the field of cell death biology.

Regulation of cell movement is mediated by stretch-activated calcium channels.

Intracellular calcium regulates many of the molecular processes that are essential for cell movement. It is required for the production of actomyosin-based contractile forces, the regulation of the structure and dynamics of the actin cytoskeletons, and the formation and disassembly of cell-substratum adhesions. Calcium also serves as a second messenger in many biochemical signal-transduction pathways. However, despite the pivotal role of calcium in motile processes, it is not clear how calcium regulates overall cell movement. Here we show that transient increases in intracellular calcium, [Ca2+]i, during the locomotion of fish epithelial keratocytes, occur more frequently in cells that become temporarily 'stuck' to the substratum or when subjected to mechanical stretching. We find that calcium transients arise from the activation of stretch-activated calcium channels, which triggers an influx of extracellular calcium. In addition, the subsequent increase in [Ca2+]i is involved in detachment of the rear cell margin. Thus, we have defined a mechanism by which cells can detect and transduce mechanical forces into biochemical signals that can modulate locomotion.

Activation of Protein Phosphorylation by Oxidants in Vascular Endothelial Cells: Identification of Tyrosine Phosphorylation of Caveolin

Oxidants play a significant role in endothelial cell dysfunction through modulation of diverse biochemical reactions and signal transduction pathways. Towards understanding the role of oxidants in vascular injury, we studied the effect of hydrogen peroxide (H 2O 2), vanadate, and pervanadate (V 4+-OOH) on [ 32P i] uptake and protein phosphorylation in bovine pulmonary artery endothelial cells (BPAEC). The incorporation of labelled [ 32P i] into BPAEC was dependent on the concentration of the oxidant employed and time of incubation. Of the oxidants tested, pervanadate (10 μM) induced maximum incorporation of [ 32P i] into cells (two- to threefold over control) followed by H 2O 2 (1 mM) and vanadate (100 μM) and clear differences in labeled protein profiles were noticed between control and oxidant treated cells. The proteins, analyzed by SDS-PAGE, showed distinct increases in labeling patterns ranging from 21–205 kDa, as evidenced by autoradiography. While the majority of the incorporated [ 32P i] was in serine/threonine residues, immunoprecipitation and immunoblotting of cell lysates, using an antiphosphotyrosine antibody, revealed that oxidant treatment resulted in significant increases in total protein tyrosine phosphorylation. Most significantly, immunoprecipitation of cell lysates, from pervanadate treatment showed distinct tyrosine phosphorylation of 22 kDa protein, which was identified as caveolin, a marker of caveolae. Pervanadate-mediated phosphorylation was effectively inhibited by staurosporine (5 μM), while genistein showed only partial attenuation. Furthermore, H 2O 2 treatment resulted in enhanced phosphorylation of 24 kDa protein, which was attenuated by genistein. In addition, oxidant-treated cells exhibited increased tyrosine kinase activity and decreased phosphatase activity. These data show differences in labeling profiles of proteins in response to different oxidants, suggesting differential modulation of distinct protein kinases/phosphatases. Copyright © 1996 Elsevier Science Inc.