Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.     

Artificial life simulation of self-assembly in bacteriophage by movable finite automata

This paper presents a model which is based on biological research using the movable finite automata (MFA) on a self-assembly of T4 phage, and exhibits the results of artificial life simulation. In the previous work, Thompson and Goel [Artificial Life, Addison Weley, 1989, pp. 317-340; Biosystems 18 (1985) 23; J. Theor. Biol. 131 (1988) 351] presented the movable finite automata (MFA) which has a capability of moving on finite automata, and simulated on a computer. They were represented individual rectangular boxes, however, the results of simulation was different from real T4 phage. We propose the sphere model as a protein structure, and simulate the self-assembly of the entire structure of the T4 phage on a computer.     

Blood-brain barrier disruption in the hypothalamus of young adult spontaneously hypertensive rats

Vascular permeability and endothelial glycocalyx were examined in young adult spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and Wistar Kyoto rats (WKY) as a control, in order to determine earlier changes in the blood-brain barrier (BBB) in the hypothalamus in chronic hypertension. These rats were injected with horseradish peroxidase (HRP) as an indicator of vascular permeability. Brain slices were developed with a chromogen and further examined with cationized ferritin, a marker for evaluating glycocalyx. Staining for HRP was seen around vessels in the hypothalamus of SHR and SHRSP, but was scarce in WKY. The reaction product of HRP appeared in the abluminal pits of endothelial cells and within the basal lamina of arterioles, showing increased vascular permeability in the hypothalamus of SHR and SHRSP, whereas there were no leaky vessels in the frontal cortex of SHR and SHRSP, or in both areas of WKY. The number of cationized ferritin particles binding to the capillary endothelial cells was decreased in the hypothalamus of SHR and SHRSP, while the number decreased in the frontal cortex of SHRSP, compared with those in WKY. Cationized ferritin binding was preserved in some leaky arterioles, while it was scarce or disappeared in other leaky vessels. These findings suggest that BBB disruption occurs in the hypothalamus of 3-month-old SHR and SHRSP, and that endothelial glycocalyx is markedly damaged there without a close relationship to the early changes in the BBB.     

Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.     

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

Cytopathologic and clinicopathologic features of ovarian hepatoid carcinoma. A case report

BACKGROUND: Hepatoid carcinoma is a rare ovarian tumor and is thought to be a different histopathologic subtype from hepatoid-type yolk sac tumor based upon its pathologic features. However, the cytopathologic characteristics of ovarian hepatoid carcinoma (OHC) have not been reported previously. We report the clinicopathologic and cytopathologic features and immunoreactivity of a case of OHC. CASE: A 36-year-old woman presented to our department with lower abdominal pain. A left ovarian tumor was found on pelvic examination, magnetic resonance imaging and computed tomography. The tumor was diagnosed as a hepatoid carcinoma of the left ovary based upon the histopathology of the surgically resected specimen. Cytopathologic specimens from a tumor touch preparation of the tumor exhibited pleomorphic tumor cells with abundant cytoplasm. The nuclei contained rough, granular chromatin and large, prominent nucleoli. Several tumor cells were multinucleated. Tumor cells were immunoreactive for alpha-fetoprotein (AFP). Hematoxylin and eosin staining revealed that the tumor cells were in a sinusoidal pattern resembling hepatocellular carcinoma without any glandular formation. The tumor cells were negative for human chorionic gonadotropin while positive for AFP, alpha-1-antitripsin, CA-125 and carcinoembryonic antigen. CONCLUSION: Cytopathologic examination is of considerable aid in the diagnosis of OHC since cytopathologic preparations highlight the characteristic cell pleomorphism.     

Reduction of phosphodiesterase 3B gene expression in peroxisome proliferator-activated receptor gamma (+/-) mice independent of adipocyte size

Phosphodiesterase 3B (PDE3B) gene expression is generally reduced in large adipocytes of obese, insulin-resistant mice. This reduced gene expression is restored by peroxisome proliferator-activated receptor (PPAR) gamma ligands accompanied by a reduced fat cell size. To determine whether PDE3B gene expression is regulated by PPAR gamma itself, we analyzed lean PPAR gamma (+/-) mice with adipocyte size comparable to control PPAR gamma (+/+) mice. In adipocytes of PPAR gamma (+/-) mice, PDE3B mRNA and protein were both reduced to 63% of wild-type levels. Basal PDE activity tended to be decreased to 70% of wild-type levels, and, similarly, insulin-induced PDE activity was significantly decreased to 70%. Thus, PPAR gamma is required for PDE3B gene expression independent of adipocyte size.