Segregation of a M404V mutation of the p62/sequestosome 1 (p62/SQSTM1) gene with polyostotic Paget's disease of bone in an Italian family

Mutations of the p62/Sequestosome 1 gene (p62/SQSTM1) account for both sporadic and familial forms of Paget's disease of bone (PDB). We originally described a methionine-->valine substitution at codon 404 (M404V) of exon 8, in the ubiquitin protein-binding domain of p62/SQSTM1 gene in an Italian PDB patient. The collection of data from the patient's pedigree provided evidence for a familial form of PDB. Extension of the genetic analysis to other relatives in this family demonstrated segregation of the M404V mutation with the polyostotic PDB phenotype and provided the identification of six asymptomatic gene carriers. DNA for mutational analysis of the exon 8 coding sequence was obtained from 22 subjects, 4 PDB patients and 18 clinically unaffected members. Of the five clinically ascertained affected members of the family, four possessed the M404V mutation and exhibited the polyostotic form of PDB, except one patient with a single X-ray-assessed skeletal localization and one with a polyostotic disease who had died several years before the DNA analysis. By both reconstitution and mutational analysis of the pedigree, six unaffected subjects were shown to bear the M404V mutation, representing potential asymptomatic gene carriers whose circulating levels of alkaline phosphatase were recently assessed as still within the normal range. Taken together, these results support a genotype-phenotype correlation between the M404V mutation in the p62/SQSTM1 gene and a polyostotic form of PDB in this family. The high penetrance of the PDB trait in this family together with the study of the asymptomatic gene carriers will allow us to confirm the proposed genotype-phenotype correlation and to evaluate the potential use of mutational analysis of the p62/SQSTM1 gene in the early detection of relatives at risk for PDB.     

Relationships of Cysteine and Lysine residues with the substrate binding site of the mitochondrial ornithine/citrulline carrier: an inhibition kinetic approach combined with the analysis of the homology structural model

To gain insights in the relationships of specific amino acid residues with the active site of the mitochondrial ornithine/citrulline carrier, we studied the effect of specific protein modifying reagents on the transport catalysed by the carrier reconstituted into liposomes. It was found that, besides the sulfhydryl reagents NEM, MTSEA, p-hydroxymercuribenzoate, diamide also the lysine reagents PLP, DIDS, SITS, the carboxyl reagents WRK, EDC and the arginine reagent methylglyoxal inhibited the carrier. NEM, MTSEA and PLP inhibited the ornithine/citrulline carrier with a completely competitive type of mechanism. A 1:1 interaction of NEM with the carrier molecule has been demonstrated. The results are in agreement with the localization of one sulfhydryl and at least one amino group in the substrate binding site. On the basis of the interferences between SH reagents and PLP in the transport inhibition, it has been deduced that the distance between the SH and the NH(2) residues of the active site should be comparable to the distance between the gamma-NH(2) and COOH residues of the ornithine molecule. The structural model of the ornithine/citrulline carrier has been obtained by homology modelling using as template the ADP/ATP carrier structure. The combined analysis of the experimental data and the structural model allows to deduce that Cys-132 is located in the substrate binding site, flanked by at least one Lys residue.     

Evolutionary conservation of Ceratitis capitata transformer gene function

Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably nonfunctional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well-conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, in this study we show that a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D. melanogaster XX; tra mutant adults. Although highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. We suggest that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects.     

Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D(-)) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.     

Potential application of low-stringency single specific primer-PCR in the identification of Leptospira in the serum of patients with suspected leptospirosis

In this study we tested the potential use of low-stringency single specific primer-PCR (LSSP-PCR) for genetically typing Leptospira directly from biological samples. Serum samples obtained from 29 patients with clinically suspected leptospirosis were amplified by specific PCR, using the previously selected G1 and G2 primers. The PCR products of approximately 300 bp were subsequently used as a template for LSSP-PCR analysis. We were able to produce genetic signatures from the leptospires present in the human samples, which permitted us to make a preliminary identification of the infective serovar by comparing the LSSP-PCR profiles obtained directly from serum samples with those from reference leptospires. Thus, LSSP-PCR has the potential to become a useful diagnostic tool for identifying leptospires in biological samples without the need for bacteria isolation and culture.     

Combined effects of radiotherapy and photodynamic therapy on an in vitro human prostate model

Human prostate cancer cells were evaluated for growth after photodynamic therapy, radiotherapy, and combined treatment. Indocyanine green was tested as a photosensitizer and radiosensitizer. Two human cell lines were used: PC-3 derived from prostate carcinoma, and EPN derived from normal prostate tissue. The light source used for the photoactivation experiments was a diode laser peaked at 805 nm. The light dose incident on cells was 108 J/cm(2). Ionizing radiation was produced by a linear accelerator, and the dose was 2, 4 and 6 Gy. Cytotoxicity was evaluated by measuring the colony forming ability of cells. Our results show that indocyanine green induces cell death by photoactivation, but it does not act as a radiosensitizer if used with ionizing radiation. The combined treatment of photodynamic therapy and radiotherapy produces an additive effect which does not depend on the sequence of the two treatments. Combined treatments could be more useful since they allow the reduction of the ionizing radiation dose to obtain the same effect as one obtainable by radiotherapy alone.     

Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.     

Optimization of recombinant human nerve growth factor production in the psychrophilic Pseudoalteromonas haloplanktis

The optimization of production strategy is a very useful tool to attain high level of recombinant protein at a low cost. A promising biotechnological application of psychrophilic bacteria is their use as non-conventional host for the recombinant production of useful proteins. The lowering of the expression temperature can in fact facilitate the correct folding of heterologous proteins that accumulate in insoluble form as inclusion bodies when produced in Escherichia coli. An example of such "difficult" proteins is the human nerve growth factor (hNGF). The gene encoding the mature form of hNGF was expressed in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 at 4 degrees C. Western blotting experiments demonstrated that the protein was produced in soluble form and translocated in the periplasmic space. Furthermore, an analytical gel filtration chromatography confirmed that the recombinant protein was largely in dimeric form. For a more efficient recombinant rhNGF production, the influence of cultivation operational strategies and growth conditions (medium composition, temperature, specific growth rate) on biomass yield and recombinant protein production was investigated in batch and chemostat cultivations. The highest product yield of soluble rhNGF (7.5mg(NGF)g(dryweight)(-1)) has been achieved in batch culture at 4 degrees C on Schatz medium with addition of tryptone and vitamins.     

The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function

The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene (Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx "retinal" territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.