Mandibular repositioning modulates IGFBP-3, -4, -5 and -6 expression in the mandibular condylar cartilage of young rats

Functional orthopedic appliances correct dental malocclusion partially by exerting indirect mechanical stimulus on the condylar cartilage, modulating growth and the adaptation of orofacial structures. However, the exact nature of the biological responses to this therapy is not well understood. Insulin-like growth factors I and II (IGF-I and II) are important local factors during growth and differentiation in the condylar cartilage [D. Hajjar, M.F. Santos and E.T. Kimura, Propulsive appliance stimulates the synthesis of insulin-like growth factors I and II in the mandibular condylar cartilage of young rats, Arch. Oral Biol. 48 (2003), 635-642]. The bioefficacy of IGFs at the cellular level is modulated by IGF binding proteins (IGFBP). The aim of this study was to verify the mRNA and protein expression of IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 in the condylar cartilage of young male Wistar rats that used a mandibular propulsive appliance for 3, 9, 15, 20, 30 or 35 days. For this purpose, sagittal sections of decalcified and paraffin-embedded condyles were submitted to immunohistochemistry and the condylar cartilage to RT-PCR. The control group showed a gradual increase in the protein expression of all IGFBPs, except IGFBP-4. Following use of the appliance, IGFBP-3 and IGFBP-6 expression decreased in the early stage of the treatment. At 20 days of treatment there was a decline in the IGFs and IGFBP-3, IGFBP-4 and IGFBP-5 expression and at 30 days there was a peak in the IGFs and all IGFBPs expression except for IGFBP-3 where the peak was observed in the control animals. The expression patterns of all IGFBPs in the condylar cartilage were similar. The modulation of IGFBP-3, -4, -5 and -6 expression in the condylar cartilage in response to the propulsive appliance suggests that those peptides are involved in the mandibular adaptation during this therapy.     

Identification and expression of the first nonmammalian amyloid-beta precursor-like protein APLP2 in the amphibian Xenopus laevis.

The Alzheimer's disease-linked amyloid-beta precursor protein (APP) belongs to a superfamily of proteins, which also comprises the amyloid-beta precursor-like proteins, APLP1 and APLP2. Whereas APP has been identified in both lower and higher vertebrates, thus far, APLP1 and 2 have been characterized only in human and rodents. Here we identify the first nonmammalian APLP2 protein in the South African claw-toed frog Xenopus laevis. The identity between the Xenopus and mammalian APLP2 proteins is approximately 75%, with the highest degree of conservation in a number of amino-terminal regions, the transmembrane domain and the cytoplasmic tail. Furthermore, amino acid residues known to be phosphorylated and glycosylated in mammalian APLP2 are conserved in Xenopus. The availability of the Xenopus APLP2 protein sequence allowed a phylogenetic analysis of APP superfamily members that suggested the occurrence of APP and preAPLP lineages with their separation predating the mammalian-amphibian split. As in mammals, Xenopus APLP2 mRNA was ubiquitously expressed and alternatively spliced forms were detected. However, the expression ratios between the mRNA forms in the various tissues examined were different between Xenopus and mammals, most prominently for the alternatively spliced forms containing the Kunitz protease inhibitor-coding region that were less abundantly expressed than the corresponding mammalian forms. Thus, the identification of APLP2 in Xenopus has revealed evolutionarily conserved regions that may help to delineate functionally important domains, and its overall high degree of conservation suggests an important role for this APP superfamily member.     

The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G1 Phase Progression in Proliferating Hepatocytes

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.     

Early Histological and Functional Effects of Chronic Copper Exposure in Rat Liver

Cu is an essential trace element capable of producing toxic effects in animals and man when ingested acutely or chronically in excess. Although chronic Cu exposure is increasingly recognized as a public health issue, its early effects remain largely unknown. We approached the significance of a moderate chronic Cu load in young rats to correlate early hepatic histopathological changes with functional alterations of liver cells. For this purpose, supplementation with 1200 ppm of Cu in rat food for 16 weeks was chosen. In these conditions, Cu load elicited a significant decrease in growth curves. There were mild light microscopy alterations in Cu-treated rats, although increasing intracellular Cu storage was correlated with longer Cu exposure both by histological and biochemical measurements. Ultrastructural alterations included lysosomal inclusions as well as mitochondrial and nuclear changes. Liver perfusion studies revealed higher rates of basal O₂ consumption and colloidal carbon-induced O₂ uptake in Cu-treated rats, with enhanced carbon-induced O₂/carbon uptake ratios and NF-κB DNA binding activity. These changes were time-dependent and returned to control values after 12 or16 weeks. It is concluded that subchronic Cu loading in young rats induces early hepatic morphological changes, with enhancement in Küpffer cell-dependent respiratory burst activity and NF-κB DNA binding, cellular responses that may prevent or alleviate the hepatotoxicity of the metal.     

Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component

Background

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.

Methodology/Principal Findings

NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.

Significance

The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00392015","term_id":"NCT00392015"}}NCT00392015     

Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha

Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.     

Retinoic acid effects on thyroid function of female rats

AimsRetinoic acid is widely used in dermatological treatment and thyroid cancer management; however its possible side-effects on normal thyroid function remains unknown. We aimed to determine the effects of retinoic acid on thyroid function of adult female rats.Main methodsFemale Wistar rats were treated with all-trans-retinoic acid and 13-cis retinoic acid for 14 and 28 days. Then, rats were killed and thyroid function was evaluated.Key findingsSerum T4 and thyrotropin levels remained unchanged, while serum T3 increased in animals treated with all-trans-retinoic acid for 14 days. No changes were observed in hepatic or renal type 1 iodothyronine deiodinase (D1) activities, while thyroid D1 was higher in animals treated for 14 days with all-trans-retinoic acid, which could be related to the increased serum T3 levels. 13-cis retinoic acid increased thyroid iodide uptake after 28 days. These results show effects of retinoic acid treatment on these thyroid proteins: sodium/iodide symporter and deiodinase.SignificanceRetinoic acid is able to interfere with normal thyroid function, increasing thyroid type 1 deiodinase activity, serum T3 levels and sodium/iodide symporter function. However, the effects are time- and retinoic acid isomer-dependent. Since serum thyrotropin levels did not change in any group, the effects observed are probably mediated by a direct retinoic acid effect on the normal thyroid.     

Novel immunostimulatory agent based on CpG oligodeoxynucleotide linked to the nontoxic B subunit of cholera toxin

In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-kappaB-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interestingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses.