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PubMed2000

Purification of GSK-3 by affinity chromatography on immobilized axin.

Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of beta-catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ss, beta-catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419-672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These "Axin-His6" matrices were found to selectively bind recombinant rat GSK-3 beta and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes.

Amino Acid Sequence↗
PubMed2005

Effects on cell viability.

Cannabinoids are known to control the cell survival/death decision, leading to different outcomes that depend on the nature of the target cell and its proliferative or differentiation status. Cannabinoids induce growth arrest or apoptosis in a number of transformed cells in culture. They do so by modulating key cell signalling pathways involved in the control of tumour cell fate. The best-characterised example is cannabinoid-induced apoptosis of glioma cells, which occurs via sustained ceramide accumulation, extracellular signal-regulated kinase activation and Akt inhibition. In addition, cannabinoid administration inhibits the angiogenesis and slows the growth of different types of tumours in laboratory animals. By contrast, most of the experimental evidence indicates that cannabinoids protect normal neurons and glial cells from apoptosis as induced by toxic insults such as glutamatergic overstimulation, ischaemia and oxidative damage. It is therefore very likely that cannabinoids regulate cell survival and cell death pathways differently in tumour and non-tumour cells. Regarding immune cells, cannabinoids affect proliferation and survival in a complex and still obscure manner that depends on the experimental setting. The findings reviewed here might set the basis for the use of cannabinoids in the treatment of cancer and neurodegenerative diseases.

Angiogenesis Inhibitors↗
PubMed1990

Mutant analysis approaches to understanding calcium signal transduction through calmodulin and calmodulin regulated enzymes.

An example set of site-specific mutagenesis studies of calmodulin has been discussed in terms of strategy and how the results can provide insight into the functioning of calmodulin. A set of common examples for the study of calcium binding and enzyme activation were discussed. Essentially, site-specific mutagenesis in these initial studies is a perturbation approach. From these perturbation studies, structural features can be correlated in future studies with function and mechanisms of action proposed. More importantly, the approach allows efficient testing of proposed mechanisms and further probing of the molecular aspects of the signal transduction pathways. Clearly, the key functional feature that must be addressed in future studies is how the calcium binding steps in the mechanism are coupled to the enzyme activation step, which is the final step of the calmodulin-enzyme binding mechanism.

Amino Acid Sequence↗
PubMed1998

Effect of Gibberellin Biosynthesis Inhibitors on Native Gibberellin Content, Growth and Floral Initiation in Sorghum bicolor.

CCC, uniconazol, ancymidol, prohexadione-calcium (BX-112), and CGA 163'935, which represent three groups of gibberellin (GA) biosynthesis inhibitors, were applied as a soil drench to Sorghum bicolor cultivars 58M (phyB-1, phytochrome B-deficient mutant) and 90M (phyB-2, equivalent phenotypically to wild type, PHYB, except for small differences in flowering dates). The inhibitors that block steps before GA12 (CCC, uniconazol, and ancymidol) lowered the concentrations of all endogenous early-C13alpha-hydroxylation pathway GAs found in sorghum: GA12, GA53, GA44, GA19, GA20, GA1, and GA8. In contrast, the inhibitors that block the conversion of GA20 --> GA1, (CGA 163'935 and BX-112) drastically reduced GA1 and GA8 levels, but they either did not change or caused accumulation of intermediates from GA12 to GA20. Combinations of pre-GA12 inhibitors and GA3 plus GA1 strongly reduced GAs other than GA1 and GA3. Each of these compounds inhibited shoot growth in both cultivars and delayed floral initiation in 90M. Floral initiation of 58M was also delayed by CCC, uniconazol, and ancymidol but not by CGA 163'935 and BX-112. This separation of shoot elongation from floral initiation in sorghum is novel. Both inhibition of shoot growth and delayed floral initiation were almost completely relieved by a mixture of GA3 and GA1 in both 58M and 90M. This observation, plus the much lower levels of endogenous GA3 than of GA1 observed in these experiments, implies that GA1 is the major endogenous GA active in shoot elongation. CGA 163'935 and BX-112 also failed to promote tillering in 58M, whereas inhibitors active before GA12 did so. The possibility that the GA20 --> GA1 inhibitors fail to block flowering and promote tillering in 58M because biosynthetic intermediates between GA12 and GA20 accumulate and/or because 58M is altered in GA metabolism in this same region of the biosynthetic pathway is discussed. Key Words. Gibberellin biosynthesis inhibitors-Phytochrome B-Flowering-Sorghum

Journal Article↗
PubMed2005

The NF-kappaB signaling pathway in human genetic diseases.

The nuclear factor-kappaB (NF-kappaB) signaling pathway plays a key role in inflammation, immune response, cell growth control and protection against apoptosis. Recently, it has been associated with several distinct genetic diseases that exhibit a large spectrum of dysfunction, such as skin inflammation, perturbed skin appendage development and immunodeficiencies. In this review, a summary of the pathophysiological consequences of impaired NF-kappaB activation in humans is provided with respect to the functions of the molecules which are mutated.

Animals↗
PubMed2003

Overexpression of IGBFB2 is a marker for malignant transformation in prostate epithelium.

Insulin-like growth factor binding protein 2 (IGFBP2) has been shown to be overexpressed in prostatic intraepithelial neoplasia (PIN) and invasive cancer and the serum level to parallel that of prostate-specific antigen (PSA) in prostate cancer. A combination of cDNA and tissue microarray results recently demonstrated overexpression of IGFBP2 in hormone refractory prostate cancer, indicating that the IGF system may be part of a key growth regulatory pathway in prostate cancer. The present study reexamines the immunohistochemical expression of IGFBP2 and its relationship to grade in tissue from 193 radical prostatectomy specimens from patients with localized prostate adenocarcinoma. We found a significant overexpression of IGFBP2 in all instances of PIN and in more than 90% of cancers regardless of the grade. An intense overexpression was noted in the neuroendocrine cells in normal glands as well as in cancer. The IGFBP2 expression was also analyzed in 18 cases of biopsy diagnosed prostate cancer. In all these cases, the glands interpreted as invasive cancer in hematoxylin-eosin stained sections overexpressed IGFBP2, without a significant correlation to grade. We conclude that overexpression of IGFBP2 is a powerful marker for malignant transformation in the prostate epithelium and suggest that optimized immunohistochemical detection of IGFBP2 expression may be an adjunct tool in the diagnosis of prostate cancer.

Adenocarcinoma↗
PubMed2026

Integrating genomics, multi-omics, CRISPR and speed breeding for stress-resilient vegetable legume improvement.

Vegetable legumes are nutritionally and ecologically important crops. However, their genetic improvement has not kept pace with the increasing challenges posed by climate change due to the polygenic nature of stress tolerance, narrow genetic diversity, and the persistent gap between molecular discoveries and field-level cultivar development. Although recent reviews have examined individual genomic tools or specific stress responses, a comprehensive synthesis integrating genomics-assisted breeding, multi-omics technologies, genome editing, and speed breeding within a unified crop improvement framework has been lacking. This review addresses that gap by critically evaluating how these complementary approaches can accelerate the development of stress-resilient vegetable legumes, including pea, common bean, cowpea, faba bean, cluster bean, yard-long bean, and hyacinth bean. This review synthesizes advances in QTL mapping, genome-wide association studies, transcriptomics, metabolomics, and CRISPR-based functional genomics that have identified key regulators and pathways underlying resistance to major biotic and abiotic stresses. Rather than considering these technologies independently, the review emphasizes their convergence into a systems-level breeding framework integrating genomic discovery, functional validation, predictive breeding, and accelerated generation advancement to improve breeding efficiency. Speed breeding, enabling up to seven to eight generations annually under optimized controlled-environment experimental conditions in cowpea, is discussed as a complementary strategy with genomic selection and genome editing. The review further identifies major translational bottlenecks, including transformation recalcitrance, limited genomic resources for underutilized vegetable legumes, inadequate multi-environment validation, and fragmented omics integration, and presents an integrated systems-breeding framework to bridge the gap between gene discovery and cultivar development.

Fabaceae↗
PubMed2026

Integrative analysis and experiment validation of SLC12A8 as a biomarker for the malignant transition from endometriosis to endometriosis associated ovarian cancer.

Endometriosis (EM) is a chronic inflammatory, estrogen‑dependent benign gynecological disorder. A subset of patients with EM may subsequently develop endometriosis‑associated ovarian cancer (EAOC), implying a biological continuum between these two conditions. Nevertheless, the molecular events underlying the progression from benign endometriotic lesions toward EAOC remain incompletely characterized. In this study, transcriptomic datasets retrieved from the GEO database were interrogated through differentially expressed gene screening, functional enrichment analysis, and weighted gene co‑expression network analysis (WGCNA) to identify key genes and pathways relevant to EM and EAOC. Candidate genes were further prioritized by integrating survival analysis via the Kaplan‑Meier Plotter, LASSO regression, random‑forest modeling, and CIBERSORT immune‑infiltration profiling. Loss and gain‑of‑function cellular models were established using siRNA and overexpression plasmids, and in‑vitro functional assays were performed to characterize the phenotypic effects of target genes.We identified several candidate genes associated with EM and EAOC and evaluated their discriminatory performance. Among them, SLC12A8 elevated expression across EM and EAOC tissues and exhibited moderate diagnostic capacity. Higher SLC12A8 expression was also associated with poorer prognosis in EAOC patients. In‑vitro experiments further demonstrated that SLC12A8 modulates proliferation, invasion, and migration in both EM and EAOC cell lines. Collectively, our exploratory research findings support SLC12A8 as a candidate functional mediator and potential biomarker linked to EM‑EAOC pathological progression, thereby extending the mechanistic understanding of these disorders.

Female↗
PubMed2026

The Genetic Architecture of Chronic Cough: From Sensory Hypersensitivity to Treatable Trait.

Chronic cough is a prevalent global clinical disorder with substantial quality-of-life impairment, and refractory cases remain a major unmet medical need. Cough hypersensitivity syndrome is the core pathological mechanism of chronic cough, and growing genetic evidence has confirmed that inherited susceptibility shapes cough hypersensitivity, clinical heterogeneity and therapeutic responsiveness, redefining chronic cough as a biologically mediated sensory-neural disorder rather than a non-specific secondary symptom of airway diseases. This review summarises genetic evidence for chronic cough from family-based studies, pharmacogenomics and genome-wide association studies (GWAS), revealing distinct genetic architectures of chronic dry cough and sputum production, with enrichment of sensory-neural pathway variants and key genetic loci such as replication factor C subunit 1 (RFC1) functional genomic analyses link genetic variation to vagal afferent excitability, and rare genetic neurological disorders further illuminate the neurogenic basis of cough hypersensitivity. Moreover, genetic insights identify tractable treatable traits and rationalise antitussive drug development, supporting genotype-guided patient stratification. We conclude that integrating genetic architecture into clinical phenotyping and translational research provides a critical framework for precision management of chronic cough, and future progress relies on harmonised deep phenotyping and multi-ancestry genetic studies.

RFC1 gene↗
PubMed1984

A mathematical model of the kinetics of 5-fluorouracil and its catabolites in freshly isolated rat hepatocytes.

A mathematical model for the kinetics of 5-fluorouracil (FUra) catabolism in liver cells is proposed. It is based on published data for the metabolism of FUra by isolated rat hepatocytes. The model relies on biochemical knowledge of the catabolic pathway. The key-steps are: the cellular uptake and the conversion of the unchanged drug to dihydrofluorouracil (FUH2) and subsequently to alpha-fluoro-beta-alanine (FBAL); the cellular fluxes of the 2 catabolites, FUH2 and FBAL. Water is partitioned between the extracellular and intracellular spaces. The first step is described by Michaelis-Menten kinetics and the other processes by first-order kinetics. Satisfactory fitting of the model validates these simplifications and provides values for the parameters describing the process. The model indicates that the kinetics of FUra disappearance are non linear, the Vmax of the first step being between 3.1 and 5.0 microM/min and the Km between 12 and 37 microM; the rate limiting step is the degradation of FUH2 (the major intracellular catabolite) with a rate constant of 0.1 to 0.02 min-1; the FUH2 transmembrane exchange is active; the exchange of the final catabolite FBAL is by diffusion.

Animals↗
PubMed1975

Entrainment of the rat's activity rhythm by cyclic light following lateral geniculate nucleus lesions.

The rhythmic running-wheel activity of rats is entrained by sensory input via the visual system. The retinal projection transmitting visual information essential for entrainment of the activity rhythm is unknown. Lesions of the lateral geniculate nucleus (LGN) disrupt two projections, the primary optic tract (POT) and the superior accessory optic tract (AOT-SF). It was found that activity remained entrained to a light cycle following LGN lesions. This indicates that the POT and the AOT-SF are not necessary for entrainment. The two remaining projections, the retinohypothalamic pathway and the inferior accessory optic tract (AOT-IF), are sufficient to maintain entrainment. In light of other investigations, it is suggested that the retinohypothalamic pathway is the key projection for synchrony of the activity rhythm with environmental illumination. It was also found that LGN lesions severely impaired the subjects' ability to learn a brightness discrimination.

Animals↗
PubMed1992

A novel 145 kd brain cytosolic protein reconstitutes Ca(2+)-regulated secretion in permeable neuroendocrine cells.

The regulated secretory pathway is activated by elevated cytoplasmic Ca2+; however, the components mediating Ca2+ regulation have not been identified. In semi-intact neuroendocrine cells, Ca(2+)-activated secretion is ATP- and cytosol protein-dependent. We have identified a novel brain protein, p145, as a cytosolic factor that reconstitutes Ca(2+)-activated secretion in two neuroendocrine cell types. The protein is a dimer of 145 kd subunits, exhibits Ca(2+)-dependent interaction with a hydrophobic matrix, and binds phospholipid vesicles, suggesting a membrane-associated function. A p145-specific antibody inhibits the reconstitution of Ca(2+)-activated secretion by cytosol, indicating an essential role for p145. The restricted expression of p145 in tissues exhibiting a regulated secretory pathway suggests a key role for this protein in the transduction of Ca2+ signals into vectorial membrane fusion events.

Animals↗
PubMed1994

Nerve-terminal proteins: to fuse to learn.

Transmitter release and membrane expansion involves the fusion of specialized vesicles to their target membranes. The mechanisms that regulate these fusion events might contribute to short- and long-term changes of synaptic efficiency that are associated with learning. A series of recently described protein-protein interactions has shed new light on vesicle binding to the cytoskeleton, vesicle docking to the target membranes and, finally, vesicle fusion and membrane retrieval. Specific steps in this pathway might be key sites for modulating the strength of synaptic connections that underlie the molecular basis of learning.

Animals↗
PubMedDate not supplied

Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas.

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon↗
PubMed1995

Inhibition by intrathecal prazosin but not yohimbine of fentanyl-induced muscular rigidity in the rat.

We evaluated the effect of intrathecally administered prazosin, alpha 1-adrenoceptor antagonist, or yohimbine, alpha 2-adrenoceptor antagonist, on fentanyl-induced muscular rigidity. Adult, male Sprague-Dawley rats were anesthetized with ketamine and were under mechanical ventilation. Fentanyl given intravenously (100 micrograms/kg) or microinjected into the bilateral locus coeruleus (LC) (2.5 microgram/50 nl) consistently evoked a significant increase in the electromyographic activity recorded from the sacrococcygeus dorsalis lateralis muscle. This implied muscular rigidity was appreciably antagonized by prior intrathecal (10 microliters) administration of prazosin (5 or 10 nmol), but not equimolar dose of yohimbine. These results suggest that the spinal alpha 1-adrenoceptors in the coerulospinal noradrenergic pathway play a key role in fentanyl-induced muscular rigidity.

Adrenergic alpha-Antagonists↗
PubMed1995

The regulation of arachidonate lipoxygenase metabolite formation in cells derived from intrauterine tissues.

Products of arachidonic acid (AA) metabolism via the lipoxygenase pathways may have key roles in the maintenance of pregnancy and the onset of labor. We have determined whether calcium ionophores can modulate the rate of biosynthesis within the uterus of five important arachidonate lipoxygenase metabolites, i.e. leukotriene B4 (LTB4), LTC4, 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE. Amnion, chorion, and decidual cells were isolated, grown to confluence and incubated with ionomycin. The production of LTB4, LTC4, 5-HETE, 12-HETE, and 15-HETE was determined using specific radioimmunoassays. Cell-specific, concentration-related stimulatory actions of ionomycin on 5-HETE, 12-HETE, 15-HETE, and LTC4 but not LTB4 production were found. A23187 had effects similar to ionomycin. Hence elevation of intracellular calcium levels can result in enhanced intrauterine production of arachidonate lipoxygenase metabolites that may affect pregnancy outcome.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2005

Discovering causes and cures for cancer from gene expression analysis.

Tumorigenesis is governed by a series of complex genetic and epigenetic changes. Both mechanisms can result in either the silencing or aberrant expression of messages in a cell. Gene expression profiling techniques such as the serial analysis of gene expression (SAGE) or microarray analysis can provide global overviews of these changes, as well identify key genes and pathways involved in this process. This review outlines the current roles of these techniques in cancer research, and how they may contribute to finding not only mechanisms of this disease, but potential targets for therapy.

Biomarkers, Tumor↗
PubMed2006

Cytokine regulation of MMP-9 in peripheral glia: implications for pathological processes and pain in injured nerve.

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced in Schwann cells hours after peripheral nerve injury and controls axonal degeneration and macrophage recruitment to the lesion. Here, we report a robust (90-fold) increase in MMP-9 mRNA within 24 h after rat sciatic nerve crush (1 to 60 days time-course). Using direct injection into a normal sciatic nerve, we identify the proinflammatory cytokines TNF-alpha and IL-1beta as potent regulators of MMP-9 expression (Taqman qPCR, zymography). Myelinating Schwann cells produced MMP-9 in response to cytokine injection and crush nerve injury. MMP-9 gene deletion reduced unstimulated neuropathic nociceptive behavior after one week post-crush and preserved myelin thickness by protecting myelin basic protein (MBP) from degradation, tested by Western blot and immunofluorescence. These data suggest that MMP-9 expression in peripheral nerve is controlled by key proinflammatory cytokine pathways, and that its removal protects nerve fibers from demyelination and reduces neuropathic pain after injury.

Animals↗