Characterization of a Novel Mouse Phenotype with Marked Hydrocephalus

In multigenic diseases, disorders where mutations in multiple genes affect the expressivity of the disease, genetic interactions play a major role in prevalence and phenotypic severity. While studying the genetic interactions between Pax3 and EdnrB in the melanocyte lineage, a new phenotype was noted in 80% of Pax3 mutants that we believe to be a novel murine model for hydrocephalus. Hydrocephalus, an accumulation of cerebrospinal fluid in the cranial cavity due to obstruction of flow in and out of the cavity, is one of the most common birth defects surpassing Down syndrome. Characteristic to hydrocephalus is a "domed" head appearance, expansion of the ventricles of the brain, and loss of neurons with hyperproliferation of glial cell types all three of which were seen in the mutant mice. The phenotype also consisted of craniofacial deformities coupled with skeletal defects including, but not limited to kyphosis, lordosis, and an apparent shortening of the some limbs. For the cellular analysis of the hydrocephalus phenotype, brains were removed and stained with two antibodies: Glial Fibrillary Acidic Protein (GFAP) and Neurofilament (NF), which are astrocyte- and neuron- specific respectively. A higher number of cells expressing GF AP and a lower number of cells expressing NF were seen in the mutant brain, when compared to control. For skeletal deformity analysis, affected mice skeletons were stained with Alizarin Red and Alcian Blue showing no apparent difference in ossification. Future genetic analysis of these mutant mice has the potential to identify novel gene modifiers involved in the promotion of this particular phenotype.

Understanding Ten-Eleven Translocation-2 in Hematological and Nervous Systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

Understanding ten-eleven translocation-2 in hematological and nervous systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. ^ In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. ^ In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.^

Purification, crystallization and partial characterization of the antitumour and insecticidal protein subunit from the delta-endotoxin of Bacillus thuringiensis var. thuringiensis

The antitumour protein from the α-endotoxin of Bacillus thuringiensis var. thuringiensis has been purified, crystallized and partially characterized. The same protein also shows the insecticidal activity. According to amino acid analysis it is an acidic protein with a molecular weight of approx. 13 000.

Purification, crystallization and partial characterization of the antitumour and insecticidal protein subunit from the σ-endotoxin of Bacillus thuringiensis var. thuringiensisstar, open

The antitumour protein from the α-endotoxin of Bacillus thuringiensis var. thuringiensis has been purified, crystallized and partially characterized. The same protein also shows the insecticidal activity. According to amino acid analysis it is an acidic protein with a molecular weight of approx. 13 000.

Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration

BACKGROUND: Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure.

METHODS: BALB/cJ and Cx3cr1 (GFP/+) mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis.

RESULTS: Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls.

CONCLUSIONS: Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.

Eppin: A Novel Protein Possessing Antimicrobial Properties Against Oral Pathogens

Background: Epididymal protease inhibitor (eppin) is a dual motif protein belonging to the whey acidic protein (WAP) family. Although expressed in numerous different tissues, to date, its functional characterisation is limited. It has been shown to exhibit antibacterial activity against Gram-negative bacteria (Escherichia coli) and antiprotease activity against some proteases of the serine protease family. We are interested in determining the role of eppin in innate immune defence. Objectives: This study aims to determine eppin's potential function in the innate immune response in the oral cavity by investigating the antimicrobial activity of eppin against relevant oral pathogens. Methods: Eppin was recombinantly expressed in E. coli cells and purified by immobilised metal affinity chromatography (IMAC). The antimicrobial effects of the protein were then assessed against two oral pathogens, Fusobacterium nucleatum and Candida albicans, using a double layer radial diffusion assay. Results: Eppin displayed antimicrobial activities against both oral pathogens tested and these activities were shown to be comparable to the well characterised antimicrobial peptide, LL-37. The antifungal effects of eppin were shown to be more potent than those of the human cathelicidin, LL-37. Conclusions: Eppin has been shown to possess both antibacterial and antifungal properties against oral pathogens, suggesting an important role for this protein in the innate immune response in the oral cavity. This study furthers our knowledge of the physiological role exerted by eppin and its possible role in the modulation of chronic diseases such as periodontitis and oral candidiasis.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation, Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved.

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Isolation, characterization and biological activity of acidic phospholipase A(2) isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIIB diffracted beyond 1.8 Angstrom resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 Angstrom, b = 54.2 Angstrom and c = 90.7 Angstrom. The crystal structure has been refined to a crystallographic residual of 16.1% (R-free = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

A human protein containing multiple types of protease-inhibitory modules

By using sensitive homology-search and gene-finding programs, we have found that a genomic region from the tip of the short arm of human chromosome 16 (16p13.3) encodes a putative secreted protein consisting of a domain related to the whey acidic protein (WAP) domain, a domain homologous with follistatin modules of the Kazal-domain family (FS module), an immunoglobulin-related domain (Ig domain), two tandem domains related to Kunitz-type protease inhibitor modules (KU domains), and a domain belonging to the recently defined NTR-module family (NTR domain). The gene encoding these WAP, FS, Ig, KU, and NTR modules (hereafter referred to as the WFIKKN gene) is intron-depleted—its single 1,157-bp intron splits the WAP module. The validity of our gene prediction was confirmed by sequencing a WFIKKN cDNA cloned from a lung cDNA library. Studies on the tissue-expression pattern of the WFIKKN gene have shown that the gene is expressed primarily in pancreas, kidney, liver, placenta, and lung. As to the function of the WFIKKN protein, it is noteworthy that it contains FS, WAP, and KU modules, i.e., three different module types homologous with domains frequently involved in inhibition of serine proteases. The protein also contains an NTR module, a domain type implicated in inhibition of zinc metalloproteinases of the metzincin family. On the basis of its intriguing homologies, we suggest that the WFIKKN protein is a multivalent protease inhibitor that may control the action of multiple types of serine proteases as well as metalloproteinase(s).

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

Purification and properties of β-glucosidase from a thermophilic fungus Humicola lanuginosa (Griffon and Maublanc) Bunce

An extracellular β-glucosidase (EC 3.2.1.21) has been purified to homogeneity from the culture filtrate of a thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce, using duplicating paper as the carbon source. The enzyme was purified 82-fold with a 43% yield by ion-exchange chromatography and gel filtration. The molecular weight of the protein was estimated to be 135,000 by gel filtration and 110,000 by electrophoresis. The sedimentation coefficient was 10.5 S. It was an acidic protein containing high amounts of acidic amino acid residues. It was poor in sulphur-containing amino acids. It also contained 9% carbohydrate. The enzyme activity was optimum at pH 4.5 and at 60°C. The enzyme was stable in the pH range 6–9 for 24 h at 25°C. The enzyme had similar affinities towards cellobiose and p-nitrophenyl-β-d-glucoside with Km values of 0.44 mM and 0.50 mM, respectively. The enzyme was capable of hydrolysing larchwood xylan, xylobiose and p-nitrophenyl-β-d-xyloside, though to a lesser extent. The enzyme was specific for the β-configuration and glucose moiety in the substrate.

Cloning and recombinant expression of a crustin-like gene from Chinese shrimp, Fenneropenaeus chinensis

Antimicrobial peptides or proteins (AMPs) are proved to be one of the most important humoral factors to resist pathogen infection. As an antimicrobial protein, crustin had been described in invertebrates as a component of the innate immune system. A crustin-like gene (CruFc) was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5'-RACE PCR. The full-length cDNA consists of 523 with 405 bp open reading frame encoding 134 amino acids and the deduced peptide contains a putative signal peptide of 17 amino acids. The sequence also contains a whey-acidic protein (WAP) domain at the C-terminal. Transcripts of CruFc were mainly detected in haemocytes and gill by RT-PCR analysis. In addition, another full-length cDNA named CshFc was also cloned from haemocytes of Chinese shrimp and its inferred amino acid sequence lacks the WAP-type 'four-disulfide core' domain. The fusion proteins containing CruFc and CshFc were, respectively, produced and the antimicrobial assays revealed that the recombinant CruFc could inhibit the growth of grain-positive bacteria in vitro but the recombinant CshFc could not inhibit at the same conditions. The difference of antimicrobial activity between recombinant CruFc and CshFc provides the evidence that the four-disulfide core domain of crustin may play an important role in its biological function. (c) 2006 Elsevier B.V. All rights reserved.

SLPI and elafin: multifunctional antiproteases of the WFDC family

SLPI (secretory leucoprotease inhibitor) and elafin represent the archetypal members of the WFDC [WAP (whey acidic protein) four disulfide core] family of proteins, and were originally characterized as protease inhibitors but have since been shown to possess a wider repertoire of activities. These functions include antimicrobial and immunomodulatory properties, suggesting that these proteins may play key roles in the innate immune response, and indicate the potential to develop some of these proteins as novel therapeutics. Susceptibility to host and bacterial protease cleavage may, however, limit the efficacy of recombinant protein therapies in diseases with a high protease burden such as CF (cystic fibrosis) lung disease. To overcome this problem, further refinement of the native proteins will be required to provide effective treatment strategies.