TCTP as a therapeutic target in melanoma treatment.

BACKGROUND: Translationally controlled tumour protein (TCTP) is an antiapoptotic protein highly conserved through phylogeny. Translationally controlled tumour protein overexpression was detected in several tumour types. Silencing TCTP was shown to induce tumour reversion. There is a reciprocal repression between TCTP and P53. Sertraline interacts with TCTP and decreases its cellular levels. METHODS: We evaluate the role of TCTP in melanoma using sertraline and siRNA. Cell viability, migration, and clonogenicity were assessed in human and murine melanoma cells in vitro. Sertraline was evaluated in a murine melanoma model and was compared with dacarbazine, a major chemotherapeutic agent used in melanoma treatment. RESULTS: Inhibition of TCTP levels decreases melanoma cell viability, migration, clonogenicity, and in vivo tumour growth. Human melanoma cells treated with sertraline show diminished migration properties and capacity to form colonies. Sertraline was effective in inhibiting tumour growth in a murine melanoma model; its effect was stronger when compared with dacarbazine. CONCLUSIONS: Altogether, these results indicate that sertraline could be effective against melanoma and TCTP can be a target for melanoma therapy.

Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus.

Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.

Characterization of Brown spider (Loxosceles intermedia) hemolymph: cellular and biochemical analyses.

This is the first study on the hemolymph from a spider of the Loxosceles genus. These animals are responsible for a great number of envenomation cases worldwide. Several studies on Loxosceles venoms have been published, and the knowledge about the venom and its toxins is considerable, not only regarding the biological and biochemical characterization, but also regarding structural, genetic and phylogenetic approaches. However, the literature on Loxosceles hemolymph is nonexistent. The main goal of the present study was to characterize biochemically the hemolymph content, and especially, to identify its different hemocytes. Moreover, many papers have already shown molecules whose source is the hemolymph and their very interesting activities and biomedical applications, for example, antifungal and antibacterial activities. A 2D-SDS-PAGE of brown spider hemolymph showed approximately 111 spots for pH 3-10 and 150 spots for pH 4-7. A lectin-blotting assay showed that hemolymph carbohydrate residues were similar to those found in venom. Several types of TAG and DAG phospholipids were found in the hemolymph and characterized by HPTLC and mass spectrometry. Four different hemocytes were characterized in Loxosceles intermedia hemolymph: prohemocyte, plasmatocyte, granulocyte and adipohemocyte. This paper opens new possibilities on toxinology, studying an unknown biological material, and it characterizes a source of molecules with putative biotechnological applications.

Reentrant spin-glass transition in a dilute magnet.

We perform a large-scale Monte Carlo simulation of a dilute classical Heisenberg model with ferromagnetic nearest neighbor and antiferromagnetic next-nearest neighbor interactions. We found that the model reproduces a reentrant spin-glass transition. That is, as the temperature is decreased, the magnetization increases rapidly below a certain temperature, reaches a maximum value, and then disappears at some lower temperature. The low temperature phase was suggested to be a spin-glass phase that is characterized by ferromagnetic clusters.

[Lung cancer with ground glass opacity diagnosed by transbronchial lung biopsy using an ultrathin bronchoscope and virtual bronchoscopy].

Although thoracic computed tomography (CT) screening indicated that there are many patients who have pulmonary shadow with ground glass opacity, it is sometimes difficult to obtain the appropriate specimens for histological diagnosis of such patients. We herein report a lung cancer patient with ground glass opacity who was diagnosed preoperatively by an ultrathin bronchoscope and virtual bronchoscopy. A 78-year-old female was admitted to our hospital due to bacterial pneumonia. At the admission, CT showed another abnormal small shadow in her right middle lobe. Since the shadow was not visible by fluoroscopy, we reconstructed the images of virtual bronchoscopy using the data obtained by multidetector CT. The location of the shadow was determined in the peripheral area of a dorsal branch of right B4aialpha. Then the transbronchial lung biopsy using an ultrathin bronchoscope with simultaneous CT guidance was performed. The histological findings of the biopsy specimens revealed that the shadow was highly suspicious for malignancy. Therefore, the right middle lobectomy was conducted, and the tumor was diagnosed as an adenocarcinoma. An ultrathin bronchoscope with virtual bronchoscopy is useful to diagnose a pulmonary shadow with ground glass opacity.

Overexpression of p8 is inversely correlated with apoptosis in pancreatic cancer.

A recently identified gene, p8, has cell growth-promoting activity and is strongly induced in acute pancreatitis. In this study, we detected p8 and single-stranded DNA (ssDNA) for apoptosis by immunohistochemistry in human pancreatic cancer. The p8 was overexpressed (>30% per 1000 cancer cells) in 26 of 44 (59%) pancreatic cancers, and apoptosis (ssDNA-positive cells >10% per 1000 cancer cells) was recognized in 18 of 44 (41%) pancreatic cancers. There was a significant inverse correlation between the p8 overexpression and apoptosis (P < 0.05). Moreover, the expression pattern of high p8 and low ssDNA was seen significantly more often in lower age (<65 years), in moderately or poorly differentiated cancers, and in node-positive cases (P < 0.05). The p8 expression and apoptosis were not significantly correlated with survival. These results suggest that p8 overexpression is involved in antiapoptotic activity and the biological characteristics of pancreatic cancer.

Lipomatous change in a brain metastasis from malignant pleural mesothelioma.

We present a case of brain metastasis from malignant mesothelioma with lipomatous change.

Expression of p8 in human pancreatic cancer.

The p8 gene is a recently identified gene with mitogenic activity. p8 expression is induced in acute pancreatitis, pancreatic development, and regeneration. However, the expression of p8 in pancreatic cancer is not reported. We investigated p8 expression in 72 human pancreatic tissues, including 38 pancreatic cancers (PCs), by immunohistochemistry. p8 was overexpressed (positive cells >25% in 1,000 cells) in 71% (27 of 38) of PCs, but in only 17% (3 of 18) of chronic pancreatitis cases. There was no overexpression in mucinous cystadenoma or in normal pancreas. The p8 overexpression rate in PC was significantly higher than that in other conditions (P < 0.05). Reverse transcription-PCR analysis confirmed p8 mRNA overexpression (tumor/nontumor ratio >2) in 75% (3 of 4) of PCs. p8 was overexpressed also in human pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). These results suggest that p8 is involved in the development of pancreatic cancer, reflecting its mitogenic activity.

Maternal active and passive smoking and fetal growth: A prospective study in Nagoya, Japan.

In order to examine the effect of maternal active and passive smoking on fetal growth, we carried out a population-based cohort study. A self-administered questionnaire was distributed to 15,207 women who notified their pregnancy from April, 1989 to March, 1991. A total of 7,411 mother-singleton infant pairs were analyzed in this study. Paternal smoking status and maternal hours exposed to environmental tobacco smoke (ETS) were used as indicators of passive smoking. Infants born to active smoking mothers were 96 g lighter, on an average, at birth than those born to non-smokers, and the relative risk for intrauterine growth retardation was 1.79 (95% confidence interval (CI) = 1.05-3.04) among active smoking mothers. Infants with smoking fathers weighted 11 g lighter, on an average, than those with non-smoking fathers, and mean birth weight of infants was reduced by 19 g among mothers exposed to ETS. The relative risk for intrauterine growth retardation in non-smoking pregnants with a smoking husband and those exposed to ETS was 0.95 (95% CI = 0.72-1.26) and 0.95 (95% CI = 0.71-1.26), respectively. Our findings indicated an adverse effect of maternal active smoking on fetal growth in Japanese pregnant population, but with small influence of maternal passive smoking.

Helicobacter pylori in the natural environment.

The presence of Helicobacter pylori (H. pylori) in the natural environment has been demonstrated in a number of studies. However, its route of infection into humans is unknown. To study this further, we attempted to detect H. pylori in the natural environment in a region of Japan with a high infection rate. Tap and well water and field soil samples were collected from around the residences of subjects who had participated in an epidemiological survey in 1996. Samples of water from rivers and ponds, and specimens of flies and cow faeces were collected in the region. DNA was extracted from the water, field soil and faecal samples after selective collection of H. pylori by the immunomagnetic-bead separation technique. H. pylori-specific DNA was detected in water, field soil, flies and cow faeces by nested polymerase chain reaction (PCR), and the ureA partial sequences of the PCR products were aligned. The nucleotide sequences of the samples amplified by PCR were highly homologous (96-100%) with the H. pylori sequence in the GenBank database and the H. pylori-specific DNA sequences were highly homologous with each other. These findings suggest the existence of H. pylori in the natural environment and a possible transmission route.

Malignant mesothelioma presenting as pulmonary metastasis ahead of growth of primary tumour.

A 59-year-old woman was admitted to Houju Memorial Hospital, Ishikawa, Japan, because of cough and fever on 30 March 1997. A diagnosis of pneumonia was made and she was given antibiotics. Her symptoms improved but failed to resolve completely on antibiotic therapy. On 9 September 1997, she revisited the hospital because of bodyweight loss and malaise. There was no history of exposure to asbestos. The chest roentgenogram revealed infiltrative shadows with vague and indistinct margins suggesting inflammatory processes, which were more extensive than those investigated on her last visit. One month later, a giant tumour was detected rapidly growing from the mediastinum and open biopsy was performed. The histological examination confirmed that the tumour was a malignant mesothelioma and the intrapulmonary nodules were its metastases. This is a rare case of pulmonary metastasis being present for several months before an appearance of primary mesothelioma.

Tricuspid valve endocarditis in a non-drug addict associated with peliosis hepatis.

A 31-year-old woman was admitted because of persistent remittent fever. Tricuspid valve endocarditis due to Staphylococcus aureus was identified as the cause of fever. The patient had no history of intravenous drug abuse, oral contraceptives or predisposing cardiac disease. Huge hepatomegaly was found without any signs of congestive heart failure. Liver enzyme abnormalities were not detected throughout the entire course of therapy. The liver biopsy specimen revealed peliosis hepatis. Treatment with panipenem/betamipron was successful, although recurrent septic pulmonary embolism occurred. The cause of the huge hepatomegaly encountered in the present case may be attributable to peliosis due to severe infection.

Expression of Bombyx family fungal protease inhibitor F from Bombyx mori by baculovirus vector.

Fungal protease inhibitor F (FPI-F) from silkworm hemolymph is a novel serine protease inhibitor of the Bombyx family. The cDNA of FPI-F was introduced into a baculovirus vector and a recombinant virus was isolated and plaque-purified. The protease inhibitory activities increased in the culture medium of insect cells and in the hemolymph of silkworms infected with the recombinant virus. Judged from the behavior on ion-exchange and reversed-phase chromatographies, amino acid compositions, amino-terminal sequences, and CD spectra, the recombinant FPI-F was identical with native FPI-F. Infection with the recombinant virus caused inhibition of larval development of the silkworm. However, the degree of the effect was different in two strains, Shinryukaku and Taiheichoan, indicating that the selection of the strain of silkworm is important in using the baculovirus expression system.

Effect of mutations on the intracellular localization of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin.

We have already cloned the polyhedrin genes of the wild-type strain H Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and its mutant, strain A. In this work, polyhedrin genes of mutant BmCPV strains C1 and C2 were cloned and their nucleotide sequences were determined. The polyhedrin amino acid sequences of strains C1 and C2 were compared with that of strain H. Strains C1 and C2 contained two and three sites of mutation in their polyhedrin genes, respectively. Four amino acids (249RLLV) were added at the carboxy terminus of the polyhedrin of strain A, C1 and C2 and the corresponding polyhedrin genes were introduced into a baculovirus expression vector. Intracellular localization of expressed polyhedrin as well as the morphology and localization of polyhedra were investigated by Western blot and microscopy analysis. Recombinant baculovirus containing the polyhedrin gene of strain H produced hexahedral polyhedra in both the cytoplasm and the nucleus. However, the hexahedral polyhedra of strain A were localized only in the nucleus. Normal polyhedra were not observed in cells infected with recombinant baculoviruses expressing strain C1 or C2 polyhedrin genes, but amorphous structures were found in infected cells. Results of expression of a chimaeric luciferase-containing carboxyl-terminal sequence of strain A demonstrated that this sequence was responsible for the nuclear localization. We suggest that a mutation at the carboxy terminus of BmCPV polyhedrin led to nuclear localization of polyhedrin and that several other mutations were responsible for modification of the crystallization pattern of polyhedrin.

Transovarian transmission of a foreign gene in the silkworm, Bombyx mori, by Autographa californica nuclear polyhedrosis virus.

We introduced a firefly luciferase gene, expressed under control of Drosophila heat shock protein gene promoter, into Autographa californica nuclear polyhedrosis virus (AcNPV). When the 5th instar larvae of the silkworm, Bombyx mori, were inoculated with the recombinant virus, luciferase activities were detected in the virus-infected larvae and pupae, and in the newly hatched larvae of the next generation. PCR amplification and Southern blot hybridization analysis demonstrated that the luciferase gene was transmitted through at least the F2 generation. In addition, the V-cathepsin gene, encoding a cysteine protease of AcNPV, was also detected in the DNA of all individuals of the F2 generation. These results show that AcNPV can be utilized as vector for the transovarian transmission of foreign genes in the silkworm.

Cryptococcal prostatitis in a patient with Behçet's disease treated with fluconazole.

A 55-year-old man with Behçet's disease presented acute urinary retention due to Cryptococcus neoformans infection of the prostate. The disease was localized to the prostate. The infection was successfully treated only with fluconazole. The patient remains well without evidence of systemic or local infection at 32 months.

Thymic carcinoma involving the thyroid gland: report of two cases.

Two new cases of thymic carcinoma involving the thyroid gland and manifesting as thyroid tumors are presented. One tumor occurred within the thyroid gland and the other totally involved the thyroid gland. Microscopically they showed lobular architecture separated by fibrous bands and consisted of solid islands of squamoid to spindled epithelial cells with whorl formation resembling Hassall's corpuscle and variable amounts of lymphocytes. The squamoid to spindled epithelial cells were positive for cytokeratin but negative for thyroglobulin and calcitonin and the majority of lymphocytes showed a T-cell phenotype. One of the two cases had been diagnosed initially as squamous cell carcinoma of the thyroid. This type of thyroid carcinoma is relatively rare, but it should be differentiated from other types of thyroid carcinomas, especially squamous cell carcinoma, medullary carcinoma, or anaplastic carcinoma of the thyroid.