EXPERIMENTAL PHYSIOLOGY, Issue 3 2000
Stefania Fulle
Brain-derived calcium-binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100-induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c-fos, c-jun, bax, bcl-x, p-15, p-21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L-type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100-derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L-type Ca2+ channel conductance with consequent [Ca2+]i increase which up-regulates, directly or indirectly, the expression of Fos. [source]

Two Key Proteins of the Vitamin D Endocrine System Come Into Crystal Clear Focus: Comparison of the X-ray Structures of the Nuclear Receptor for 1,,25(OH)2 Vitamin D3, the Plasma Vitamin D Binding Protein, and Their Ligands,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2003
Mathew T Mizwicki
First page of article [source]

Induction of Transcriptional Activity of the Cyclic Adenosine Monophosphate Response Element Binding Protein by Parathyroid Hormone and Epidermal Growth Factor in Osteoblastic Cells,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002
John T. Swarthout
Abstract Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3, (GSK-3,) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal,activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects. [source]

Use of combined in silico expression data and phylogenetic analysis to identify new oocyte genes encoding RNA binding proteins in the mouse

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2008
Laurence Drouilhet
Abstract During folliculogenesis, oocytes accumulate maternal mRNAs in preparation for the first steps of early embryogenesis. The processing of oocyte mRNAs is ensured by heterogeneous nuclear ribonucleoproteins (hnRNPs) genes that encode RNA binding proteins implied in mRNA biogenesis, translation, alternative splicing, nuclear exportation, and degradation. In the present work, by combining phylogenetic analyses and, when available, in silico expression data, we have identified three new oocyte-expressed genes encoding RNA binding proteins by using two strategies. Firstly, we have identified mouse orthologs of the Car1 gene, known to be involved in regulation of germ cell apoptosis in C. elegans, and of the Squid gene, required for the establishment of anteroposterior polarity in the Drosophila oocyte. Secondly, we have identified, among genes whose ESTs are highly represented in oocyte libraries, a paralog of Poly(A) binding protein,Interacting Protein 2 (Paip2) gene, known to inhibit the interaction of the Poly(A)-Binding Protein with Poly(A) tails of mRNAs. For all of these genes, the expression in oocyte was verified by in situ hybridization. Overall, this work underlines the efficiency of in silico methodologies to identify new genes involved in biological processes such as oogenesis. Mol. Reprod. Dev. 75: 1691,1700, 2008. © 2008 Wiley-Liss, Inc. [source]

ORIGINAL ARTICLE: An Interleukin-23 Binding Protein in Mid-Trimester Amniotic Fluid

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2009
Catherine Herway
Problem, The binding of mid-trimester amniotic fluid to cytokines was evaluated. Method of study, Purified tumor necrosis factor-, (TNF-,), interleukin (IL)-10, IL-12, and IL-23 were incubated with amniotic fluid from 25 women undergoing a mid-trimester amniocentesis, or with bovine serum albumin or saline, and cytokine binding to monoclonal antibodies was quantitated by ELISA. Aliquots of amniotic fluid were heated to 95°C for 15 min and then retested for IL-23 binding. The effect of amniotic fluid dilution on IL-23 quantitation was evaluated. Results, All amniotic fluids had a negligible effect on TNF-,, IL-10, and IL-12 detection. In marked contrast, pre-incubation with amniotic fluid from each subject reduced the subsequent ability to detect IL-23 by >50%. The extent of inhibition was directly proportional to the amniotic fluid dilution and was markedly reduced following heating at 95°C for 15 min. Amniotic fluids from White, Black, Asian, East Indian, and Hispanic women were equally effective. Conclusion, Interleukin-23 and IL-12 share a common p40 subunit and no inhibition of IL-12 was apparent. It appeared that a component of mid-trimester amniotic fluid specifically interacts with the p19 subunit unique to IL-23. Mid-trimester amniotic fluid reactivity with IL-23 may be a mechanism to limit intra-amniotic neutrophil-derived inflammation. [source]

The auxin-binding protein 1 is essential for the control of cell cycle

THE PLANT JOURNAL, Issue 2 2007
Karine M. David
Summary The phytohormone auxin has been known for >50 years to be required for entry into the cell cycle. Despite the critical effects exerted by auxin on the control of cell division, the molecular mechanism by which auxin controls this pathway is poorly understood, and how auxin is perceived upstream of any change in the cell cycle is unknown. Auxin Binding Protein 1 (ABP1) is considered to be a candidate auxin receptor, triggering early modification of ion fluxes across the plasma membrane in response to auxin. ABP1 has also been proposed to mediate auxin-dependent cell expansion, and is essential for early embryonic development. We investigated whether ABP1 has a role in the cell cycle. Functional inactivation of ABP1 in the model plant cell system BY2 was achieved through cellular immunization via the conditional expression of a single-chain fragment variable (scFv). This scFv was derived from a well characterized anti-ABP1 monoclonal antibody previously shown to block the activity of the protein. We demonstrate that functional inactivation of ABP1 results in cell-cycle arrest, and provide evidence that ABP1 plays a critical role in regulation of the cell cycle by acting at both the G1/S and G2/M checkpoints. We conclude that ABP1 is essential for the auxin control of cell division and is likely to constitute the first step of the auxin-signalling pathway mediating auxin effects on the cell cycle. [source]

Preclinical Studies with Adrenomedullin and Its Binding Protein as Cardiovascular Protective Agents for Hemorrhagic Shock

CARDIOVASCULAR THERAPEUTICS, Issue 3-4 2006
Rongqian Wu
ABSTRACT Traumatic injury is a major, largely unrecognized public health problem in the US that cuts across race, gender, age, and economic boundaries. The resulting loss of productive life years exceeds that of any other disease, with societal costs of $469 billion annually. Most trauma deaths result either from insufficient tissue perfusion due to excessive blood loss, or the development of inflammation, infection, and vital organ damage following resuscitation. Clinical management of hemorrhagic shock relies on massive and rapid infusion of fluids to maintain blood pressure. However, the majority of victims with severe blood loss do not respond well to fluid restoration. The development of effective strategies for resuscitation of traumatic blood loss is therefore critically needed. We have recently discovered that the vascular responsiveness to a recently-discovered potent vasodilatory peptide, adrenomedullin (AM) is depressed after severe blood loss, which may be due to downregulation of a novel specific binding protein, AM binding protein-1 (AMBP-1). Using three different animal models of hemorrhage (controlled hemorrhage with large volume resuscitation, controlled hemorrhage with low volume resuscitation, and uncontrolled hemorrhage with minimum resuscitation), we have shown that cell and organ injury occurs after hemorrhage despite fluid resuscitation. Administration of AM/AMBP-1 significantly improves cardiac output, heart performance and tissue perfusion, attenuates hepatic and renal injury, decreases pro-inflammatory cytokines, prevents metabolic acidosis, and reduces hemorrhage-induced mortality. Thus, administration of AM/AMBP-1 appears to be a novel and useful approach for restoring cardiovascular responses, preventing organ injury, and reducing mortality after hemorrhagic shock. [source]

Disulfide Bond Substitution by Directed Evolution in an Engineered Binding Protein

CHEMBIOCHEM, Issue 8 2009
Antoine Drevelle Dr.
Abstract Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications. The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques. [source]

Penicillin Binding Proteins: key players in bacterial cell cycle and drug resistance processes

FEMS MICROBIOLOGY REVIEWS, Issue 5 2006
Pauline Macheboeuf
Abstract Bacterial cell division and daughter cell formation are complex mechanisms whose details are orchestrated by at least a dozen different proteins. Penicillin-binding proteins (PBPs), membrane-associated macromolecules which play key roles in the cell wall synthesis process, have been exploited for over 70 years as the targets of the highly successful ,-lactam antibiotics. The increasing incidence of ,-lactam resistant microorganisms, coupled to progress made in genomics, genetics and immunofluorescence microscopy techniques, have encouraged the intensive study of PBPs from a variety of bacterial species. In addition, the recent publication of high-resolution structures of PBPs from pathogenic organisms have shed light on the complex intertwining of drug resistance and cell division processes. In this review, we discuss structural, functional and biological features of such enzymes which, albeit having initially been identified several decades ago, are now being aggressively pursued as highly attractive targets for the development of novel antibiotherapies. [source]

Serum Insulin-Like Growth Factor-1 Binding Proteins 1 and 2 and Mortality in Older Adults: The Health, Aging, and Body Composition Study

JOURNAL OF AMERICAN GERIATRICS SOCIETY, Issue 7 2009
Donglei Hu PhD
OBJECTIVE: To evaluate the relationship between serum insulin-like growth factor 1 (IGF-1), IGF-1 binding protein 1 (IGFBP-1), and IGF-1 binding protein 2 (IGFBP-2) and fasting insulin, fasting glucose, adiposity, and mortality in older adults. DESIGN: A prospective cohort study with mean follow-up of 6.2 years. SETTING: Participants were recruited and followed at two centers affiliated with academic medical institutions. PARTICIPANTS: Six hundred twenty-five men and women aged 70 and older and in good health at the time of enrollment. MEASUREMENTS: Serum IGF-1, IGFBP-1, and IGFBP-2; fasting serum insulin; fasting serum glucose; visceral fat; and total percent fat. RESULTS: Higher IGFBP-1 and higher IGFBP-2 were significantly associated with lower fasting insulin, lower fasting glucose, and lower adiposity, but higher IGFBP-1 and IGFBP-2 were associated with greater mortality. In multivariate adjusted models, the hazard ratio for all-cause mortality was 1.48 (95% confidence interval (CI)=1.14,1.92) per standard deviation (SD) increase in IGFBP-2 and 1.34 (95% CI=1.01,1.76) per SD increase in IGFBP-1. No association was found between IGF-1 and all-cause mortality. CONCLUSIONS: Higher IGFBP-1 and IGFBP-2 are associated with lower adiposity and decreased glucose tolearance but also with greater all-cause mortality. Higher levels of serum IGF-1 binding protein (IGFBP) may indicate greater IGF-1 activity and thus represent an association between higher IGF-1 activity and mortality in humans. [source]

Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein

CHEMBIOCHEM, Issue 6 2005
Torun Engfeldt
Abstract Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-naphthalenesulfonic acid (EDANS) and 6-(7-nitrobenzofurazan-4-ylamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA. [source]

Genetic and plasma variation of insulin-like growth factor binding proteins in relation to prostate cancer incidence and survival

THE PROSTATE, Issue 12 2009
Mattias Johansson
Abstract BACKGROUND Binding proteins regulate bioavailability of insulin-like growth factor-I (IGF-I) in the circulation and affect apoptosis of tumor cells in the prostate. We analyzed genetic variation within genes coding for IGF binding proteins in relation to prostate cancer incidence and survival. We also investigated if circulating IGFBP3 affects prostate cancer-specific survival. MATERIALS AND METHODS Eleven haplotype tagging SNPs and two single SNPs in the IGFBP1, IGFBP3, and IGFALS genes were genotyped within the CAncer Prostate in Sweden (CAPS) study including 2,774 cases and 1,736 controls. Plasma samples for analyses of total- and intact IGFBP3 levels were available for 1,521 cases and 909 controls. Complete follow-up of vital status was achieved by linkage to the Swedish Cause of Death Register. RESULTS We found no clear association between the genetic variants and prostate cancer incidence or survival. The rare allele of the IGFBP3 SNP rs2854744 was associated with elevated plasma levels of total IGFBP3 (Ptrend,=,9,×,10,8), but not intact IGFBP3 (Ptrend,=,0.16). Elevated levels of total- (Ptrend,=,0.03) and intact IGFBP3 (Ptrend,=,6,×,10,14) were associated with increased risk of prostate cancer specific death. Treatment and tumor characteristics accounted for the association with total IGFBP3, whereas the association with intact IGFBP3 was attenuated, but still statistically significant in adjusted analysis (Ptrend-adjusted,=,0.0004). Elevated intact IGFBP3 was also significantly associated with increased risk of prostate cancer-specific death among patients who were chemically or surgically castrated (Ptrend-adjusted,=,0.0003), and among patients who had not been treated (Ptrend-adjusted,=,0.02). CONCLUSIONS Circulating levels of intact IGFBP3 measured after diagnosis is associated with increased risk of prostate cancer-specific death. Prostate 69:1281,1291, 2009. © 2009 Wiley-Liss, Inc. [source]

Reduced alpha adrenergic mediated contraction of renal preglomerular blood vessels as a function of gender and aging

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2005
John C. Passmore
Abstract As human males age, a decline in baroreflex-mediated elevation of blood pressure occurs due, at least in part, to a reduction in alpha-1 adrenergic vasoconstrictor function. Alpha adrenergic constriction is mediated by guanosine triphosphate binding Protein (G Protein) coupled signaling pathways. Alpha-1 A/C, B, and D adrenergic receptor expressions, measured by GeneChip array, are not reduced during aging in renal blood vessels of male or female rats. Alpha-1 A GeneChip expression is greater, at all ages studied, in females than in males. Prazosin binding by alpha-1 adrenergic receptors is greater in young adult female rats than in young adult male rats; however, it is reduced with aging in both male and female rats. G alpha q GeneChip expression declines while expression of adrenergic receptor kinase (GRK2) and tyrosine phosphatases (TyrP) increase with aging in male rats. The declines in alpha-1 adrenergic receptor binding and G alpha q expression and also the increases in GRK2 and TyrP expression likely relate to the age-related decline of vasoconstriction in male rats. The information that the expression of alpha-1 A adrenergic receptors is greater in female rats and (GRK2) expression does not increase during aging could relate to the gender differences in vasoconstrictor function with aging. Gene therapy to ameliorate the age-related decline in renal function could possibly reduce the need for renal dialysis. Signaling pathways such as those reviewed herein may provide an outline of the molecular pathways needed to move toward successful renal gene therapy for aging individuals. J. Cell. Biochem. © 2005 Wiley-Liss, Inc. [source]

Androgen replacement therapy improves function in male rat muscles independently of hypertrophy and activation of the Akt/mTOR pathway

ACTA PHYSIOLOGICA, Issue 4 2009
C. Hourdé
Abstract Aim:, We analysed the effect of physiological doses of androgens following orchidectomy on skeletal muscle and bone of male rats, as well as the relationships between muscle performance, hypertrophy and the Akt/mammalian target of rapamycin (mTOR) signalling pathway involved in the control of anabolic and catabolic muscle metabolism. Methods:, We studied the soleus muscle and tibia from intact rats (SHAM), orchidectomized rats treated for 3 months with vehicle (ORX), nandrolone decanoate (NAN) or dihydrotestosterone (DHT). Results:, Orchidectomy had very little effect on the soleus muscle. However, maximal force production by soleus muscle (+69%) and fatigue resistance (+35%) in NAN rats were both increased when compared with ORX rats. In contrast, DHT treatment did not improve muscle function. The relative number of muscle fibres expressing slow myosin heavy chain and citrate synthase activity were not different in NAN and ORX rats. Moreover, NAN and DHT treatments did not modify muscle weights and cross-sectional area of muscle fibres. Furthermore, phosphorylation levels of downstream targets of the Akt/mTOR signalling pathway, Akt, ribosomal protein S6 and eukaryotic initiation factor 4E-binding protein 1 were similar in muscles of NAN, DHT and ORX rats. In addition, trabecular tibia from NAN and DHT rats displayed higher bone mineral density and bone volume when compared with ORX rats. Only in NAN rats was this associated with increased bone resistance to fracture. Conclusion:, Physiological doses of androgens are beneficial to muscle performance in orchidectomized rats without relationship to muscle and fibre hypertrophy and activation of the Akt/mTOR signalling pathway. Taken together our data clearly indicate that the activity of androgens on muscle and bone could participate in the global improvement of musculoskeletal status in the context of androgen deprivation induced by ageing. [source]

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

CYTOSKELETON, Issue 8 2006
Masaya Morita
Abstract Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10,6 M Ca2+. Motility features changed when Ca2+ concentrations were increased from 10,6 to 10,5 M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca2+. Thus, it is possible that Ca2+ binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca2+ -binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by 45Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca2+ -binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca2+/CaM-dependent manner for regulating the dynein activity. A 32P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca2+/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10,5 M Ca2+. It is possible that an increase in Ca2+ induces Ca2+/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source]

Nucleolar colocalization of TAF1 and testis-specific TAFs during Drosophila spermatogenesis

DEVELOPMENTAL DYNAMICS, Issue 10 2007
Chad E. Metcalf
Abstract In Drosophila, testis-specific TBP-associated factors (tTAFs) predominantly localize to spermatocyte nucleoli and regulate the transcription of genes necessary for spermatocyte entry into meiosis. tTAFs are paralogs of generally expressed TAF subunits of transcription factor IID (TFIID). Our recent observation that the generally expressed TAF1 isoform TAF1-2 is greatly enriched in testes prompted us to explore the functional relationship between general TAFs and tTAFs during spermatogenesis. Analysis by immunofluorescence microscopy revealed that among the general TFIID subunits examined (TATA-box binding protein [TBP], TAF1, TAF4, TAF5, and TAF9), only TAF1 colocalized with the tTAF Mia in spermatocyte nucleoli. Nucleolar localization of TAF1, but not Mia, was disrupted in tTAF mutant flies, and TAF1 dissociated from DNA prior to Mia as spermatocytes entered meiosis. Taken together, our results suggest stepwise assembly of a testis-specific TFIID complex (tTFIID) whereby a TAF1 isoform, presumably TAF1-2, is recruited to a core subassembly of tTAFs in spermatocyte nucleoli. Developmental Dynamics 236:2836,2843, 2007. © 2007 Wiley-Liss, Inc. [source]

Sexual dimorphism of g-protein subunit Gng13 expression in the cortical region of the developing mouse ovary

DEVELOPMENTAL DYNAMICS, Issue 7 2007
Akihiro Fujino
Abstract In our search for genes required for the development and function of mouse gonads, we identified Gng13 (guanine nucleotide binding protein 13, gamma), a gene with an embryonic expression pattern highly restricted to the ovary. Based on reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, Gng13 is expressed in both XX and XY gonads at embryonic day (E) 11.5, but becomes up-regulated in the XX gonad by E12.5. Expression is retained after treatment with busulfan, a chemical known to eliminate germ cells, pointing to the soma as a site of Gng13 transcription. In situ hybridization of embryonic ovarian tissue sections further localized the expression to the cortex of the developing XX gonad. Gng13 expression in the adult is also highly restricted. Northern blot analyses and Genomic Institute of the Novartis Research Foundation expression profiling of adult tissues detected very high expression in the cerebrum and cerebellum, in addition to, a weaker signal in the ovary. Gng13 belongs to a well-known family of signal transduction molecules with functions in many aspects of development and organ physiology. Here, we report that, in the developing mouse embryo, expression of Gng13 mRNA is highly restricted to the cortex of the XX gonad during sexual differentiation, suggesting a role for this gene during ovarian development. Developmental Dynamics 236:1991,1996, 2007. © 2007 Wiley-Liss, Inc. [source]

Epidermal transient down-regulation of retinol - binding protein 4 and mirror expression of apolipoprotein Eb and estrogen receptor 2a during zebrafish fin and scale development

DEVELOPMENTAL DYNAMICS, Issue 11 2006
Angèle Tingaud-Sequeira
Abstract Very little is known about the molecular control of skin patterning and scale morphogenesis in teleost fish. We have found radially symmetrical epidermal placodes with down-regulation of retinol - binding protein 4 (rbp4) expression during the initial paired fin and scale morphogenesis in zebrafish (Danio rerio). This finding may be related to changes in keratinocyte cytodifferentiation and/or the integument retinoid metabolism. rbp4 transcripts are expressed afterward in the central epidermis of the scale papilla and gradually extend to the epidermis, covering the growing scale, whereas no transcripts were detected in posterior margin epidermis. In contrast, induction of apolipoprotein Eb (apoeb) and up-regulation of estrogen receptor 2a (esr2a) transcripts were observed in the epidermis at initiator sites of zebrafish ectodermal/dermal appendage morphogenesis. This expression was maintained in the posterior margin epidermis of the formed scales. esr2a was also strongly expressed in neuromasts, whereas no rbp4 and apoeb transcripts were detected in these mechanosensory structures. The observed epidermal molecular events suggest that epidermis patterning is due to an activator,inhibitor mechanism operational at epidermal,dermal interaction sites. rbp4 transcript expression was also strongly down-regulated by 1-phenyl-2-thio-urea (PTU). As this inhibitor is commonly used to block obscuring pigmentation during in situ hybridization studies, this finding suggests that PTU should be used with caution, particularly in studying skin development. Developmental Dynamics 235:3071,3079, 2006. © 2006 Wiley-Liss, Inc. [source]

Retinoids and spermatogenesis: Lessons from mutant mice lacking the plasma retinol binding protein

DEVELOPMENTAL DYNAMICS, Issue 6 2006
Norbert B. Ghyselinck
Abstract Using Rbp4 -null mice as models, we have established for the first time the kinetics of the spermatogenetic alterations during vitamin A deficiency (VAD). Our data demonstrate that the VAD-induced testicular degeneration arises through the normal maturation of germ cells in a context of spermatogonia differentiation arrest. They indicate that retinoic acid (RA) appears dispensable for the transition of premeiotic to meiotic spermatocytes, meiosis, and spermiogenesis. They confirm that RA plays critical roles in controlling spermatogonia differentiation, spermatid adhesion to Sertoli cells, and spermiation, and suggest that the VAD-induced arrest of spermatogonia differentiation results from simultaneous blocks in RA-dependent events mediated by RA receptor , (RAR,) in spermatogonia and by RAR, in Sertoli cells. They also provide evidence that expression of major RA-metabolizing enzymes is increased in mouse Sertoli cells upon VAD and that vitamin A-deficient A spermatogonia differ from their RA-sufficient counterparts by the expression of the Stra8 gene. Developmental Dynamics 235:1608,1622, 2006. © 2006 Wiley-Liss, Inc. [source]

Level of purposeful hand function as a marker of clinical severity in Rett syndrome

DEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 9 2010
JENNY DOWNS
Aim, We investigated relationships between hand function and genotype and aspects of phenotype in Rett syndrome. Method, Video assessment in naturalistic settings was supplemented by parent-reported data in a cross-sectional study of 144 females with a mean age of 14 years 10 months (SD 7y 10mo; range 2y,31y 10mo), 110 of whom had a mutation of the methyl CpG binding protein 2 (MECP2) gene. Ordinal logistic regression was used to assess relationships between hand function and MECP2 mutation, age, a modified Kerr score, Functional Independence Measure for Children (WeeFIM), ambulation level, and frequency of hand stereotypies. Results, Approximately two-thirds of participants demonstrated purposeful hand function, ranging from simple grasping skills to picking up and manipulating small objects. In participants with a confirmed MECP2 mutation, those with the p.R168X mutation had the poorest hand function on multivariate analysis with C-terminal deletion as the baseline (odds ratio [OR] 0.19; 95% confidence interval [CI] 0.04,0.95), whereas those with the p.R133C or p.R294X mutation had better hand function. Participants aged 19 years or older had lower hand function than those aged less than 8 years (OR 0.36; 95% CI 0.14,0.92). Factors that were associated with better hand function were lower Kerr scores for a 1-point increase in score (OR 0.77; 95% CI 0.69,0.86), higher WeeFIM scores for a 1-point increase in score (OR 1.08; 95% CI 1.04,1.12), and greater ambulation than those completely dependent on carers for mobility (OR 22.64; 95% CI 7.02,73.08). The results for participants with a confirmed pathogenic mutation were similar to results obtained when participants without a mutation were also included. Interpretation, Our novel assessment of hand function in Rett syndrome correlated well with known profiles of common MECP2 mutations and overall clinical severity. This promising assessment could measure clinical responses to therapy. [source]

The mode of action of thiazolidinediones,

DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue S2 2002
Hans Hauner
Abstract The thiazolidinediones (TZDs) or ,glitazones' are a new class of oral antidiabetic drugs that improve metabolic control in patients with type 2 diabetes through the improvement of insulin sensitivity. TZDs exert their antidiabetic effects through a mechanism that involves activation of the gamma isoform of the peroxisome proliferator-activated receptor (PPAR,), a nuclear receptor. TZD-induced activation of PPAR, alters the transcription of several genes involved in glucose and lipid metabolism and energy balance, including those that code for lipoprotein lipase, fatty acid transporter protein, adipocyte fatty acid binding protein, fatty acyl-CoA synthase, malic enzyme, glucokinase and the GLUT4 glucose transporter. TZDs reduce insulin resistance in adipose tissue, muscle and the liver. However, PPAR, is predominantly expressed in adipose tissue. It is possible that the effect of TZDs on insulin resistance in muscle and liver is promoted via endocrine signalling from adipocytes. Potential signalling factors include free fatty acids (FFA) (well-known mediators of insulin resistance linked to obesity) or adipocyte-derived tumour necrosis factor-, (TNF-,), which is overexpressed in obesity and insulin resistance. Although there are still many unknowns about the mechanism of action of TZDs in type 2 diabetes, it is clear that these agents have the potential to benefit the full ,insulin resistance syndrome' associated with the disease. Therefore, TZDs may also have potential benefits on the secondary complications of type 2 diabetes, such as cardiovascular disease. Copyright © 2002 John Wiley & Sons, Ltd. [source]

Effect of RBP4 gene variants on circulating RBP4 concentration and Type 2 diabetes in a Chinese population

DIABETIC MEDICINE, Issue 1 2008
C. Hu
Abstract Aims Retinol binding protein 4 (RBP4) is a newly discovered adipokine, which plays a role in insulin resistance and obesity. The aim of this study was to determine the relationship between genetic variants of the RBP4 gene, circulating RBP4 concentrations and phenotypes related to glucose and lipid metabolism in the Chinese population. Methods We sequenced exons and the putative promoter region to identify single nucleotide polymorphisms (SNPs) in the RBP4 gene in 32 Chinese subjects. Additional SNPs were selected from a public database to increase marker density. Taking account of the pairwise linkage disequilibrium and minor allele frequencies, a subset of SNPs was further genotyped in 255 Type 2 diabetic patients and 372 normal control subjects. Circulating RBP4 concentrations and phenotypes related to glucose and lipid metabolism were measured. Results Ten SNPs were identified and five were further genotyped in the full sample. No individual SNP was significantly associated with Type 2 diabetes, but a rare haplotype CAA formed by +5388 C>T, +8201 T>A and +8204 T>A was more frequent in diabetic patients (P = 0.0343, empirical P = 0.0659 on 10 000 permutations). In both groups, non-coding SNPs were associated with circulating RBP4 concentrations (P < 0.05). In the normal control subjects, the SNP +5388 C>T was associated with serum C-peptide levels both fasting and 2 h after an oral glucose tolerance test (P = 0.0162 and P = 0.0075, respectively). Conclusion Our findings suggest that genetic variants in the RBP4 gene may be associated with circulating RBP4 concentration and phenotypes related to glucose metabolism. [source]

crabp and maf highlight the novelty of the amphioxus club-shaped gland

ACTA ZOOLOGICA, Issue 2 2004
William R. Jackman
Abstract The club-shaped gland (csg) is a prominent organ during the development of amphioxus. However, the evolutionary significance of this pharyngeal structure has been a mystery because of the lack of an obvious corollary in vertebrates or other close relatives. To address the homology of the csg by molecular means, we report the cloning and expression patterns of two amphioxus genes expressed during its development, crabp and maf. Amphioxus maf is a bzip transcription factor expressed early in csg formation in the forming of the ventral duct of the gland. crabp encodes a cellular retinoic acid binding protein and is expressed widely in the csg later in its development. We compare these genes to the expression of AmphiKrox, a zinc-finger transcription factor previously reported to be expressed during csg development. Together these genes mark different spatial and temporal aspects of csg formation. However, we find little evidence to suggest homology of the csg with other organs in amphioxus or other chordates. We therefore propose that the csg can be viewed as an evolutionary novelty that probably arose within the amphioxus lineage. [source]

Measurement of dissociation rate of biomolecular complexes using CE

ELECTROPHORESIS, Issue 3 2009
Peilin Yang
Abstract Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide,protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of,53,s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30,cm long. When using Fyn-SH2-SH3 tagged with glutathione S -transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7,s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0±2.7 and 58.8±6.1,s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that although all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions. [source]

Molecular analysis of the phosphorus starvation response in Trichodesmium spp.

ENVIRONMENTAL MICROBIOLOGY, Issue 9 2009
Elizabeth D. Orchard
Summary The marine diazotroph Trichodesmium is a major contributor to primary production and nitrogen fixation in the tropical and subtropical oceans. These regions are often characterized by low phosphorus (P) concentrations, and P starvation of Trichodesmium could limit growth, and potentially constrain nitrogen fixation. To better understand how this genus responds to P starvation we examined four genes involved in P acquisition: two copies of a high-affinity phosphate binding protein (pstS and sphX) and two putative alkaline phosphatases (phoA and phoX). Sequence analysis of these genes among cultured species of Trichodesmium (T. tenue, T. erythraeum, T. thiebautii and T. spiralis) showed that they all are present and conserved within the genus. In T. erythraeum IMS101, the expression of sphX, phoA and phoX were sensitive to P supply whereas pstS was not. The induction of alkaline phosphatase activity corresponded with phoA and phoX expression, but enzyme activity persisted after the expression of these genes returned to basal levels. Additionally, nifH (nitrogenase reductase; involved in nitrogen fixation) expression was downregulated under P starvation conditions. These data highlight molecular level responses to low P and lay a foundation for better understanding the dynamics of Trichodesmium P physiology in low-P environments. [source]

Urinary L-FABP and anaemia: distinct roles of urinary markers in type 2 diabetes

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2 2010
M. Von Eynatten
Eur J Clin Invest 2010; 40 (2): 95,102 Abstract Background, Urinary liver-type fatty acid binding protein (L-FABP) and kidney injury molecule (KIM)-1, novel urinary biomarkers of renal tubulointerstitial function, have previously been associated with acute ischaemic kidney injury. We studied the clinical significance of urinary L-FABP, KIM-1 and N -acetyl-,-glucosaminidase (NAG) as potential markers of renal function and chronic ischaemic injury in patients with diabetic nephropathy. Material and methods, A total of 130 type 2 diabetes patients with early diabetic nephropathy and 40 healthy controls were studied. Urinary L-FABP, KIM-1, NAG, albumin excretion rate (AER) and creatinine clearance were obtained from 24-h urine samples, and correlated with measures of red blood cell count, renal function and metabolic control. Results, Urinary L-FABP was significantly increased in diabetes patients compared with healthy controls [8·1 (interquartile 0·6,11·6) vs. 2·4 (0·5,3·6) ,g/g creatinine, P < 0·001] and correlated with AER (r = 0·276, P = 0·002), creatinine clearance (r = ,0·189, P = 0·033) and haemoglobin levels (r = ,0·190, P = 0·030). In multivariable linear regression analysis, haemoglobin (, = ,0·247, P = 0·015) and AER (, = 0·198, P = 0·046) were significant predictors of urinary L-FABP. Prevalent anaemia was independently associated with a 6-fold risk for increased tubulointerstitial kidney damage (upper vs. lower two L-FABP tertiles: OR, 6·06; 95% CI: 1·65,22·23; P = 0·007). Urinary KIM-1 was not significantly associated with kidney function, AER, or measures of red blood cell count while urinary NAG was associated with parameters of glucose control and renal function. Conclusions, Different urinary biomarkers may reflect distinct pathophysiological mechanisms of tubulointerstitial damage in early diabetic nephropathy: Urinary L-FABP could be a novel biomarker for chronic intrarenal ischaemia. [source]

Retinol binding protein isolated from acute renal failure patients inhibits polymorphonuclear leucocyte functions

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2004
G. Cohen
Abstract Background, Protein factors accumulating in sera of patients with end-stage renal disease (ESRD) that interfere with the nonspecific immune response by inhibiting essential functions of polymorphonuclear leucocytes (PMNLs) have previously been described. No such factor has been isolated from acute renal failure (ARF) patients to date. Materials and methods, Using a three-step chromatographic procedure involving ion exchange, size exclusion and hydrophobic interaction chromatography we purified the apo- and holo-form of retinol binding protein (RBP) from high-flux dialyser (polyacrylonitrile; AN69) ultrafiltrates of patients with ARF. Their effect on the chemotaxis of PMNLs isolated from healthy donors was determined by the under-agarose method. Whole-blood assays applying flow cytometry were used to assess phagocytosis and the oxidative metabolism of PMNLs. Apoptosis was assessed by determining the DNA content using propidium iodide. Results, Isolated apo- and holo-forms of RBP were truncated on their C-terminus as determined by mass spectrometry. All isolates significantly inhibited the chemotactic movement of PMNLs obtained from healthy donors and the PMNL oxidative metabolism stimulated by E. coli. These effects were concentration dependent. Retinol binding protein had no influence on the PMNL oxidative metabolism stimulated by PMA and on PMNL phagocytosis. Commercially available RBP isolated from urine influenced PMNL functions in the same way. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 significantly attenuated the phagocytosis-induced respiratory burst and RBP did not lead to a further decrease. Polymorphonuclear leucocyte apoptosis was significantly inhibited by RBP. Conclusions, The apo- and holo-forms of RBP isolated from the ultrafiltrate of ARF patients inhibit PMNL chemotaxis, oxidative metabolism and apoptosis. Therefore, RBP may be considered a uraemic toxin contributing to a disturbed immune defence. [source]

Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2008
Fumihiko Mouri
Abstract High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1, protein. The ,131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S -transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes. [source]

Constitutive expression of the FK506 binding protein 51 (FKBP51) in bone marrow cells and megakaryocytes derived from idiopathic myelofibrosis and non-neoplastic haematopoiesis

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2004
Oliver Bock
Abstract: Objectives:, Overexpression of FK506 binding protein 51 (FKBP51) in megakaryocytic progenitor cells generated from purified CD34+ cells in patients with idiopathic myelofibrosis (IMF) has been demonstrated. It has been suggested that FKBP51 is involved in the dysregulation of the apoptotic programme with consecutive prolongation of cell survival. The knowledge of FKBP51 and its expression in bone marrow cells and mature megakaryocytes in non-neoplastic haematopoiesis and IMF is sparse. Methods:, To evaluate a potential overexpression of FKBP51 in patients with IMF (n = 37) compared with non-neoplastic haematopoiesis (n = 31), total bone marrow cells as well as single megakaryocytes, isolated by laser microdissection, were quantitatively analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). By applying immunohistochemistry, FKBP51 gene expression was correlated with staining pattern and cellular localisation of the corresponding FKBP51 protein. Results:, We demonstrated that FKBP51 is constitutively expressed in non-neoplastic haematopoiesis. FKBP51 gene expression by total bone marrow cells as well as megakaryocytes was not significantly different in IMF. FKBP51 protein expression could be localised to myeloid progenitor cells as well as megakaryocytes. In particular, megakaryocytes were stained almost exclusively nuclear for FKBP51. No differences in expression patterns between both IMF and control cases could be demonstrated. Conclusions:, For the first time, FKBP51 expression, in particular gene expression and subcellular localization was described in bone marrow cells of non-neoplastic and neoplastic haematopoiesis grown in vivo. We conclude that FKBP51 could be temporarily overexpressed in megakaryocytic progenitors rather than contribute to the accumulation of mature megakaryocytes in IMF. [source]

The Drosophila PRR GNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll -pathway activation function

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2010
Alexey A. Matskevich
Abstract The Drosophila Toll -signaling pathway controls the systemic antifungal host response. Gram-negative binding protein 3 (GNBP3), a member of the ,-glucan recognition protein family senses fungal infections and activates this pathway. A second detection system perceives the activity of proteolytic fungal virulence factors and redundantly activates Toll. GNBP3hades mutant flies succumb more rapidly to Candida albicans and to entomopathogenic fungal infections than WT flies, despite normal triggering of the Toll pathway via the virulence detection system. These observations suggest that GNBP3 triggers antifungal defenses that are not dependent on activation of the Toll pathway. Here, we show that GNBP3 agglutinates fungal cells. Furthermore, it can activate melanization in a Toll -independent manner. Melanization is likely to be an essential defense against some fungal infections given that the entomopathogenic fungus Beauveria bassiana inhibits the activity of the main melanization enzymes, the phenol oxidases. Finally, we show that GNBP3 assembles "attack complexes", which comprise phenoloxidase and the necrotic serpin. We propose that Drosophila GNBP3 targets fungi immediately at the inception of the infection by bringing effector molecules in direct contact with the invading microorganisms. [source]