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PubMed1998

The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity.

Cyclin-dependent kinase 5 (Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. Here we show that the p35/Cdk5 kinase co-localizes with Rac in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth.

Animals↗

PubMed2003

The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin.

Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.

3T3 Cells↗

PubMed2007

Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia.

Aberrant activation of beta-catenin promotes cell proliferation and initiates colorectal tumorigenesis. However, the expansion of tumours and the inadequacy of their local vasculature results in areas of hypoxia where cell growth is typically constrained. Here, we report a novel diversion in beta-catenin signalling triggered by hypoxia. We show that hypoxia inhibits beta-catenin-T-cell factor-4 (TCF-4) complex formation and transcriptional activity, resulting in a G1 arrest that involves the c-Myc-p21 axis. Additionally, we find that hypoxia inducible factor-1alpha (HIF-1alpha) competes with TCF-4 for direct binding to beta-catenin. DNA-protein interaction studies reveal that beta-catenin-HIF-1alpha interaction occurs at the promoter region of HIF-1 target genes. Furthermore, rigorous analyses indicate that beta-catenin can enhance HIF-1-mediated transcription, thereby promoting cell survival and adaptation to hypoxia. These findings demonstrate a dynamic role for beta-catenin in colorectal tumorigenesis, where a functional switch is instigated to meet the ever-changing needs of the tumour. This study highlights the importance of the microenvironment in transcriptional regulation.

Adaptation, Physiological↗

PubMed2004

Evidence for two distinct binding sites for tau on microtubules.

The microtubule-associated protein tau regulates diverse and essential microtubule functions, from the nucleation and promotion of microtubule polymerization to the regulation of microtubule polarity and dynamics, as well as the spacing and bundling of axonal microtubules. Thermodynamic studies show that tau interacts with microtubules in the low- to mid-nanomolar range, implying moderate binding affinity. At the same time, it is well established that microtubule-bound tau does not undergo exchange with the bulk medium readily, suggesting that the tau-microtubule interaction is essentially irreversible. Given this dilemma, we investigated the mechanism of interaction between tau and microtubules in kinetic detail. Stopped-flow kinetic analysis reveals moderate binding affinity between tau and preassembled microtubules and rapid dissociation/association kinetics. In contrast, when microtubules are generated by copolymerization of tubulin and tau, a distinct population of microtubule-bound tau is observed, the binding of which seems irreversible. We propose that reversible binding occurs between tau and the surface of preassembled microtubules, whereas irreversible binding results when tau is coassembled with tubulin into a tau-microtubule copolymer. Because the latter is expected to be physiologically relevant, its characterization is of central importance.

Binding Sites↗

PubMed2004

Cell cycle-mediated regulation of smooth muscle alpha-actin gene transcription in fibroblasts and vascular smooth muscle cells involves multiple adenovirus E1A-interacting cofactors.

Expression of smooth muscle alpha-actin in growth factor-induced myofibroblasts and in differentiated vascular smooth muscle cells is transcriptionally controlled by multiple positive or negative trans-acting factors interacting with distinct cis-elements in the 5'-flanking region of the gene. Because none of the transcriptional regulators reported to date is smooth muscle cell- or myofibroblast-specific per se, the dynamic interplay among many factors interacting at specific sites along the promoter appears to be a signature feature of smooth muscle alpha-actin gene regulation in these cell types. Herein, the ability of the adenovirus E1A 12 S protein to bind and functionally inactivate specific cell regulatory factors has been exploited to identify several previously unknown coactivators of the mouse smooth muscle alpha-actin promoter in rodent fibroblasts and vascular smooth muscle cells. In transient cotransfection assays, ectopic expression of wild type E1A suppressed promoter activity in a dose- and cis-element-dependent manner. In asynchronous cells, N-terminal E1A mutants defective in CREB-binding protein (CBP) and p300 binding capacity exhibited markedly reduced inhibitory activity toward a smooth muscle alpha-actin promoter driven by a composite TEF-1-, SRF-, and Sp1/3-regulated enhancer. In synchronized cells, however, a more complex mutant E1A inhibitory pattern indicated that collaboration between CBP/p300 and the retinoblastoma family of pocket proteins was required to produce a fully functional enhancer. Cotransfection experiments conducted with Rb(-/-) fibroblasts demonstrated the necessity of pRB in augmenting smooth muscle alpha-actin enhancer/promoter activity. Physical interaction studies with the use of purified wild type and mutant E1A proteins confirmed that CBP, p300, and pRB were targets of E1A binding in nuclear extracts of vascular smooth muscle cells and/or fibroblasts. Collectively, these results suggest that a repertoire of E1A-interacting proteins, namely CBP/p300 and pRB, serve to integrate the activities of multiple trans-acting factors to control smooth muscle alpha-actin gene transcription in a cell type- and cell cycle-dependent manner.

Actins↗

PubMed2004

Calcium-dependent regulation of the cell cycle via a novel MAPK--NF-kappaB pathway in Swiss 3T3 cells.

Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation and transformation. We investigated the role of the serum-induced intracellular calcium increase in the NF-kappaB--dependent cell cycle progression in Swiss 3T3 fibroblasts. Noninvasive photoactivation of a calcium chelator (Diazo-2) was used to specifically disrupt the transient rise in calcium induced by serum stimulation of starved Swiss 3T3 cells. The serum-induced intracellular calcium peak was essential for subsequent NF-kappaB activation (measured by real-time imaging of the dynamic p65 and IkappaBalpha fluorescent fusion proteins), cyclin D1 (CD1) promoter-directed transcription (measured by real-time luminescence imaging of CD1 promoter-directed firefly luciferase activity), and progression to cell division. We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent. Inhibition of the MAPK- but not the PtdIns3K-dependent pathway inhibited NF-kappaB signaling, and further, CD1 transcription and cell cycle progression. These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK--NF-kappaB pathway in Swiss 3T3 cells.

Animals↗

PubMed1992

Detection, characterization, and bioavailability of membrane-associated iron in the intact sickle red cell.

It is hypothesized that membrane-associated iron in the sickle red cell is of pathophysiologic importance, but the actual existence of such iron in the intact cell has been questioned. Using a strategy whereby membrane iron can be detected through its bioavailability for catalyzing peroxidation, we used phospholipid exchange protein to load membranes of intact erythrocytes (RBC) with approximately 2% phosphatidylethanolamine hydroperoxide (PEOOH) and monitored the development of peroxidation by-products during subsequent incubation. Normal RBC loaded with PEOOH developed very little peroxidation, but vitamin E-replete sickle RBC showed an exuberant peroxidation response that was not seen in cells loaded with control nonoxidized phosphatidylethanolamine. Ancillary studies of sickle RBC revealed that the catalytic iron included both heme iron and free iron located at the bilayer inner leaflet. Significantly, these studies also revealed that peroxidation after PEOOH loading is promoted by cellular dehydration and inhibited by hydration, thus identifying a dynamic interaction between hemoglobin (sickle >> normal) and membrane lipid. High-reticulocyte control RBC and sickle trait RBC behaved exactly like normal RBC, while HbCC RBC and RBC having membranes gilded with hemoglobin iron because of prior exposure to acetylphenylhydrazine showed an abnormal peroxidation response like that of sickle RBC. Indeed, the peroxidation response of RBC loaded with PEOOH paralleled amounts of iron measured on inside-out membranes prepared from them (r = 0.783, P < 0.01). These studies corroborate existence of membrane-associated heme and free iron in the intact sickle cell, and they document its bioavailability for participation in injurious peroxidative processes. That association of cytosolic sickle hemoglobin with membrane lipid is modulated by cell hydration status provides a mechanism that may help explain increased development of oxidative membrane lesions in abnormally dehydrated sickle RBC regardless of the mechanism underlying their formation.

Anemia, Sickle Cell↗

PubMedDate not supplied

In search of the architect of learning: a commentary on scaffolding as a metaphor for instructional interactions.

In this article, I accept Stone's invitation to reevaluate the metaphor of scaffolding, building on his helpful analysis. Specifically, I identify what has been appealing about the scaffolding metaphor by describing how, as an intervention researcher working with students with learning disabilities, I have used the metaphor to describe a specific instructional approach in my own research. Then, I revisit and critique scaffolding as an instructional metaphor. I also comment on Stone's assertion that a more explicit description of communicative interchanges is required to more clearly describe instructional scaffolding. Finally, I conclude that the scaffolding metaphor is fundamentally flawed and advance an alternative view that may more successfully promote correspondence between instructional activities and our emerging vision of the dynamics of teaching and learning.

Child↗

PubMed2004

Spontaneous growth hormone (GH) secretion is not directly affected by ghrelin in either short normal prepubertal children or children with GH neurosecretory dysfunction.

Ghrelin, a specific endogenous ligand for the GH secretagogue receptor, stimulates GH secretion in humans when given in pharmacological amounts. Under physiological conditions, however, it is controversial whether ghrelin affects GH secretion and vice versa. No studies have reported on the relationship between daily ghrelin and GH secretion in children. Therefore, plasma ghrelin and GH concentrations over a 24-h period were studied in 10 prepubertal short normal children (five females and five males) to determine the potential relationship between the secretion of these two hormones. Furthermore, five prepubertal patients (two females and three males) with GH neurosecretory dysfunction (GHNSD) were studied in the same way to assess potential alterations in ghrelin secretion in a condition associated with distinct GH changes. No gender difference in ghrelin spontaneous secretion was detected in either short normal children or GHNSD patients, and in both male and female subjects, ghrelin was secreted in a pulsatile and circadian fashion, with a nocturnal surge. Twenty-four-hour secretion and daytime ghrelin secretion of short normal children were similar to those in GHNSD patients, whereas nighttime hormone secretion in the latter group was significantly greater than that in short normal children. The cross-correlation of 24-h ghrelin and GH levels revealed significant positive and negative correlations, which were similar in the two groups examined. The positive one, with GH leading ghrelin, might reflect a somatostatin (SMS)-mediated inhibitory effect on both GH and ghrelin secretion (low SMS levels are followed by high GH and ghrelin levels, and vice versa). The negative correlation, with ghrelin leading GH, might again reflect the positive effect of ghrelin on SMS, as shown in both animal and human studies. In conclusion, the results of the present study indicate that ghrelin secretion in prepubertal children is pulsatile and is not sexually dimorphic. Although the parallelism of ghrelin and GH dynamics hints at the potential relevance of endogenous ghrelin as a promoter of GH release, our data do not support this hypothesis. We suggest that the interactions of ghrelin and GH are the result of SMS action. SMS inhibits GH secretion not only by a direct effect on the pituitary and by inhibiting hypothalamic GHRH, but also through the suppression of ghrelin release.

Body Height↗

PubMed2002

Tau and axonopathy in neurodegenerative disorders.

The microtubule (MT)-associated protein (MAP) tau in neurons has been implicated as a significant factor in the axonal growth, development of neuronal polarity, and the maintenance of MT dynamics. Tau is localized to the axon, and is known to promote MT assembly and to stabilize axonal MTs. These functions of tau are primarily regulated by the activities of protein kinases and phosphatases. In Alzheimer's disease and other neurodegenerative disorders, abundant filamentous tau inclusions are found to be major neuropathological characteristics of these diseases. Both somato-dendritic and axonal tau lesions appear to be closely associated with axonal disruption. Furthermore, recent discoveries of pathogenic mutations on the tau gene suggest that abnormalities of tau alone are causative of neurodegeneration. Finally, analyses of transgenic mice that express human tau proteins have enabled in vivo quantitative assessments of axonal functions and have provided information about mechanistic relationships between pathological alteration of tau and axonal degeneration.

Animals↗

PubMed2004

Brain-derived neurotrophic factor regulation of retinal growth cone filopodial dynamics is mediated through actin depolymerizing factor/cofilin.

The molecular mechanisms by which neurotrophins regulate growth cone motility are not well understood. This study investigated the signaling involved in transducing BDNF-induced increases of filopodial dynamics. Our results indicate that BDNF regulates filopodial length and number through a Rho kinase-dependent mechanism. Additionally, actin depolymerizing factor (ADF)/cofilin activity is necessary and sufficient to transduce the effects of BDNF. Our data indicate that activation of ADF/cofilin mimics the effects of BDNF on filopodial dynamics, whereas ADF/cofilin inactivity blocks the effects of BDNF. Furthermore, BDNF promotes the activation of ADF/cofilin by reducing the phosphorylation of ADF/cofilin. Although inhibition of myosin II also enhances filopodial length, our results indicate that BDNF signaling is independent of myosin II activity and that the two pathways result in additive effects on filopodial length. Thus, filopodial extension is regulated by at least two independent mechanisms. The BDNF-dependent pathway works via regulation of ADF/cofilin, independently of myosin II activity.

14-3-3 Proteins↗

PubMed1999

The 35UZ transposon of Drosophila melanogaster reveals differences in maintenance of transcriptional control between embryonic and larval stages.

The D. melanogaster transposon P[35UZ] contains a lacZ reporter gene fused to 35 kb of Ubx upstream sequences which drive a Ubx-like expression in embryos and in metathoracic imaginal discs. Transposition of P[35UZ] followed by analysis of different lines in wild-type and mutant backgrounds allowed us to analyze the interplay between Ubx regulatory elements, including the Polycomb response element (PRE), located inside the transposon and cis-acting regulatory elements, located outside. We found that all lines show a Ubx-like beta-galactosidase expression pattern in the embryo, but proximity to strong imaginal enhancers can change this pattern drastically. These data illustrate how maintenance of gene expression depends on the chromosomal environment and on dynamic interactions between all developmentally regulated enhancers located close to a promoter under PcG control.

Animals↗

PubMed2001

Vascular architecture of normal human urinary bladder and its remodeling in cancer, as revealed by corrosion casting.

The vasculature of normal human urinary bladder wall and its tumors were studied using corrosion casting and scanning electron microscopy. In the normal wall, a regular organization of highly tortuous horizontal and vertical vessels allows accommodation of the vascular system to spatial changes resulting from the filling/voiding cycle. The vasculature of tumors is a result of remodeling of preexisting wall vessels associated with gradual growth of the neoplastic tissue. The shape and arrangement of blood vessels in different regions of the tumor seem to reflect a variable dynamics of tumor development and possible influence of various angiogenesis-promoting growth factors.

Blood Vessels↗

PubMed2003

[E-commerce of pharmaceuticals].

The emergence of the Internet as a new communications and information technology caused major social and cultural changes. The dramatic increase in accessibility and availability of information empowered the consumer by closing the information gap between the consumer and different suppliers. The objective of this article is to review many new internet-supported applications related to the pharmaceutical market. E-commerce is divided into two major components: Business to Consumer (B to C), and Business to Business (B to B). The main applications in B to C are dissemination of medical and drug information, and the sale of drugs through the Internet. Medical information on the Internet is vast and very helpful for patients, however, its reliability is not guaranteed. Online pharmacies increase the accessibility and availability of drugs. Nevertheless, several obstacles such as security of the data provided (both financial and clinical) prevent the widespread use of online pharmacies. Another risk is the health authorities' inability to regulate Internet sites effectively. Therefore, unregulated sale of prescription drugs, fake or substandard, often occurs on the Internet. B to B relates to physicians, clinics, hospitals, HMO's and pharmaceutical companies. There is a vast number of applications ranging from clinical research, marketing and sales promotion, to drug distribution and logistics. In conclusion, the Internet is dynamic and has contributed to the development of numerous new applications in the field of pharmaceuticals. Regulatory authorities should be active in developing new policies that will deal with those new Internet-based applications.

Drug Industry↗

PubMed2003

Hepatic differentiation from embryonic stem cells in vitro.

OBJECTIVE: To investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepatocyte for hepatocyte replacement therapy in the treatment of liver failure. METHODS: ES cells from Balb/C mice were cultured and maintained in an undifferentiated state in gelatin-coated dishes using Dulbecco's modified Eagle's medium (DMEM) containing 1000 U/ml leukemia inhibitory factor (LIF). Then, LIF was withdrawn from the DMEM to allow the ES cells to develop into embryonic bodies (EBs). EBs were plated onto tissue culture dishes, and growth factors such as acidicfibroblast growth factor (aFGF) and hepatocyte growth factor (HGF) were added to the medium to promote directional differentiation. The course of development and differentiation was observed dynamically using an inversion microscope. The expression of hepatic proteins, such as alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK8), cytokeratin 18 (CK18), in cytoplasm was analyzed by immunocytochemistry (ICC). The concentration of ALB in the medium was determined dynamically by radioimmunoassay (RIA). RESULTS: ES cells replicated as clones, without differentiating, in DMEM containing LIF. They developed into EBs in medium without LIF. Our ICC assay showed that differentiating cells did not express hepatic proteins, such as AFP, ALB, CK8, and CK18 until day 7, day 9, day 11, and day 11, respectively (up to 2 days later when growth factors are not present). The concentration of AFP in the medium was first detected on day 8, at a concentration of 3.4 ng/ml, and increased to 22.8 ng/ml by day 15. The concentration of ALB in the medium was 0.2 micro g/ml on day 11, and increased to 2.2 micro g/ml by day 15. ALB-positive cells under ICC manifest morphological structures were consistent with normal mouse hepatocytes. The differentiation ratio of hepatocytes in the ES cell differentiation system was 30% on day 15 (significantly lower without the presence of growth factors). CONCLUSIONS: ES cells can differentiate into mature hepatocytes. Growth factors, such as aFGF and HGF, can enhance this differentiation and produce sufficient numbers of functional hepatocytes. This method may be a reliable new way of differentiating ES cells into hepatocytes for use in replacement therapy in the treatment of liver failure.

Animals↗

PubMed1991

[A return to callus healing using dynamic osteosynthesis procedures. Principles, indications, complications, x-ray diagnosis of interlocking nailing and dynamic monofixators in comparison with conventional bone plates].

A disproportionately high rate of complications such as osteomyelitis, nonunion and refracture after plating have brought about a change in our understanding of the process of fracture healing. Locked nailing and dynamic external Monofixators, however, avoid fracture exposure obviate the necessity of arranging fragments and aim at callus healing of fractures. Fracture hematoma and axial micromovements of these dynamic stabilizing devices are two of the yet known elements to promote callus production.

Bone Nails↗

PubMed1987

[The main trends in the area of the pharmacokinetic study of chemotherapeutic agents].

Development of rational antibiotic dosing is mainly limited by the absence of a clear conception of the minimum efficient concentration. Studies on antimicrobial effect kinetics in dynamic models in vitro simulating pharmacokinetic profiles observed in humans markedly promote the problem solution. Such an approach enabled one to reveal a relationship between duration or intensity of the antibiotic effect and the area under the concentration/time curve. This relationship depends on the antibiotic pharmacokinetic profile. It is advisable to evaluate antibiotic efficacy against microorganisms by the minimum efficient area under the concentration/time curve and not by the antibiotic minimum efficient concentration. Thus, it is possible to estimate simultaneous contribution of two factors to the antimicrobial effect: drug concentration and exposure time.

Anti-Bacterial Agents↗

PubMed1997

[Knemometry in the assessment of short term effects of growth hormone therapy in children with several growth disorders].

BACKGROUND: Knemometer is a non invasive device for measuring the leg length and evaluating statural variations otherwise not disclosed by means of Harpenden stadiometer. In several clinical conditions of short stature, knemometer allows to evaluate the short-term effect of growth promoting agents, as well as the role of infections on growth dynamics. METHODS: The short-term (37-56 days) variability of the leg length in subjects with isolated growth hormone deficiency (1 case), Turner syndrome (2 cases) and Silver-Russel syndrome (1 case) treated with rhGH have been evaluated. The measurements were performed weekly by the same operator, at the same weekday and daytime. RESULTS AND CONCLUSIONS: The results show a correlation between intercurrent illnesses and growth failure, as well as a growth resumption at the end of the former, despite the etiology of short stature. Growth hormone treatment seems to exert an influence on growth only in the subject with GH deficiency, whereas a similar short term effect is not found in the cases of Turner syndrome and Silver Russel syndrome. This fact suggests that, also by knemometry, a most prolonged follow-up is required to evaluate the effect of GH on short term growth.

Adolescent↗