Effects of fluoride on expression of bone-specific genes in developing Xenopus laevis larvae.

The effect of fluoride treatment on the expression of a panel of osteogenic and stress markers in Stage 55 premetamorphic Xenopus larvae was examined at the precise onset of replacement of the larval cartilaginous skeleton with bone. A dosing regimen of 10 mmol/L sodium fluoride over 8 days was followed, during which time larvae developed to Stage 58, when the process of progressive ossification takes place in the vertebral column and membranous bones of the skull, pelvic, and pectoral girdles and portions of the appendicular skeleton. Markers of bone formation, including COL1A1, the transcription factors Osterix, RUNX2-II, and matrix metalloproteinases MMP1 and MMP13, decreased relative to age-matched controls, though the osteoblast marker BGLAP was not significantly altered.

Parameters for effective in vitro production of zinc finger nucleic acid-binding proteins.

Immobilized metal affinity chromatography (IMAC) is widely used for the production of recombinant proteins for a variety of applications; however, a number of challenges are typically encountered by researchers depending on the properties of the specific proteins in question. Here, we describe technical issues we have encountered in production of recombinant zinc finger nucleic acid-binding proteins by IMAC intended for detailed and accurate in vitro analysis. The process encountered leading to a modified IMAC protocol for effective production of high-purity, native zinc finger nucleic acid-binding proteins is described in detail.

Expression of hsp90 alpha and hsp90 beta during Xenopus laevis embryonic development.

Background: Members of the eukaryotic Hsp90 family function as important molecular chaperones in the assembly, folding and activation of cellular signaling in development. Two hsp90 genes, hsp90 and hsp90, have been identified in fish and homeothermic vertebrates but not in poikilothermic vertebrates. In the present study, the expression of hsp90 and hsp90 genes in Xenopus laevis, which is phylogenetically positioned between zebrafish and mammals, has been addressed.

Modulation of Hsf1 activity by novobiocin and geldanamycin.

Since Hsp90 is a known modulator of HSF1 activity, we examined the effects of two pharmacological inhibitors of Hsp90, novobiocin and geldanamycin, on HSF1 DNA-binding activity in the Xenopus oocyte model system. Novobiocin exhibits antiproliferative activity in culture cells and interacts with a C-terminal ATP-binding pocket on Hsp90, inhibiting Hsp90 autophosphorylation. Treatment of oocytes with novobiocin followed by heat shock results in a dose-dependent decrease in HSF1 DNA-binding and transcriptional activity.

A comparison of Hsp90alpha and Hsp90beta interactions with cochaperones and substrates.

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions.

Biochemical characterization of Yin Yang 1-RNA complexes.

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated.

Assembly of the Yin Yang 1 transcription factor into messenger ribonucleoprotein particles requires direct RNA binding activity.

The early stages of vertebrate development depend heavily on control of maternally transcribed mRNAs that are stored for long periods in complexes termed messenger ribonucleoprotein particles (mRNPs) and utilized selectively following maturation and fertilization. The transcription factor Yin Yang 1 (YY1) is associated with cytoplasmic mRNPs in vertebrate oocytes; however, the mechanism by which any of the mRNP proteins associate with mRNA in the oocyte is unknown. Here we demonstrate the mechanism by which YY1 associates with mRNPs depends on its direct RNA binding activity.

Asymmetric distribution of metals in the Xenopus laevis oocyte: a synchrotron X-ray fluorescence microprobe study.

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte.

Heat shock factor 1 is required for constitutive Hsp70 expression and normal lens development in embryonic zebrafish.

Heat shock factors (HSFs) are the major transcription factors responsible for heat-induced upregulation of heat shock protein (Hsp) genes. All three mammalian HSFs (HSF1, HSF2, HSF4) have also been shown to be required for normal mammalian development. It is currently unknown if HSFs play similarly important roles during normal development of non-mammalian vertebrates.

Protein phosphatase 5 is a negative modulator of heat shock factor 1.

The major stress protein transcription factor, heat shock factor (HSF1), is tightly regulated through a multilayered activation-deactivation process involving oligomerization, post-translational modification, and interaction with the heat shock protein (Hsp90)-containing multichaperone complex. Conditions of proteotoxic stress, such as heat shock, trigger reversible assembly of latent HSF1 monomers into DNA-binding homotrimers that bind with high affinity to cognate heat shock elements. Transactivation is a second and independently regulated function of HSF1 that is accompanied by hyperphosphorylation and appears to involve a number of signaling events.

Actin expression is induced and three isoforms are differentially expressed during germination in Zea mays.

Previous analysis of actin in a dicotyledonous plant, Phaseolus vulgaris (or common bean), showed very low actin levels in cotyledons but they were concentrated in the embryo axis. Upon imbibition, actin expression increased 5-fold and a maximum of four actin isoforms were observed, two of them transient and two major ones were steadily expressed. In this work, analysis of the actin expression in a monocotyledonous plant, Zea mays (or maize), and over a longer period of germination/growth, showed that striking similarities exist.

Troglitazone reduces heat shock protein 70 content in primary rat hepatocytes by a ubiquitin proteasome independent mechanism.

Troglitazone (TRG) is an antidiabetic agent that increases the insulin sensitivity of target tissues in non-insulin-dependent diabetes mellitus. Therapy with troglitazone has been associated with severe hepatic injury in a small percentage of patients and the mechanism of TRG-induced hepatotoxicity remains unclear. A family of highly conserved stress proteins identified as heat shock proteins (Hsps), are well-known to protect cells against a wide variety of toxic conditions such as extreme temperature changes, oxidative stress and toxic drugs.

l-carnosine and verapamil inhibit hypoxia-induced expression of hypoxia inducible factor (HIF-1 alpha) in H9c2 cardiomyoblasts.

Contractile failure of myocardial cells is a common cause of mortality in ischemic heart disease. In response to hypoxic conditions, cells upregulate the activity of hypoxia-inducible factor 1 (HIF-1) and express a number of genes encoding proteins that either enhance O (2)delivery or increase cellular ATP levels. HIF-1 is a heterodimer of bHLH-PAS proteins, HIF-1 alpha and HIF-1 beta.

The Yin Yang 1 transcription factor associates with ribonucleoprotein (mRNP) complexes in the cytoplasm of Xenopus oocytes.

Yin Yang 1 (YY1) is a multifunctional transcription factor that activates, represses, or initiates transcription of a diverse assortment of genes. Previous studies suggest a role for YY1 in cellular growth and differentiation, but its biological function during development of the vertebrate oocyte or embryo remains to be determined. We recently showed that YY1 is abundantly expressed throughout oogenesis and early embryonic stages of Xenopus, but it is sequestered in the cytoplasm and does not function directly in transcriptional regulation.