Production and purification of recombinant somatolactin beta and its effects on melanosome aggregation in zebrafish

A second form of somatolactin, somatolactin beta (SLbeta), was recently discovered in zebrafish (Danio rerio). This novel subtype of somatolactin is distantly related to somatolactin alpha (SLalpha) found in teleost species and is produced in a different region of the pituitary. To date, no physiological study of SLbeta has been reported. In order to study the physiological functions of SLbeta, recombinant SLbeta protein has been produced and purified. The cDNA of zebrafish SLbeta was cloned into a pET100 bacteria expression vector and His-tagged fusion proteins were produced in BL21 (DE3) Escherichia coli cells. The majority of recombinant somatolactins produced by E. coli were isolated in inclusion bodies although a small percentage of recombinant proteins (<1%) were also found in soluble form. Fusion proteins were solubilized from inclusion bodies using 6M guanidine hydrochloride. Pure recombinant somatolactins were obtained by affinity purification. The estimated molecular weight of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis correlates with the molecular mass calculated from the deduced amino acid sequence of SLbeta. Thereafter, specific polyclonal antibodies against the recombinant SLbeta were developed. These antibodies recognized specifically a group of cells located in the anterior pars intermedia of the pituitary. The antibodies did not react with SLalpha, growth hormone or prolactin cells in the zebrafish pituitary glands. Furthermore, recombinant SLbeta induced melanosome aggregation in a concentration-dependent manner in skin of zebrafish scales. Significant melanosome aggregation was observed in zebrafish melanophores at a concentration of 1 microg/ml. These results, combined with previous reports demonstrate that the recombinant SLbeta proteins produced here are bioactive. The function of inducing melanosome aggregation is conserved among the somatolactin functions.     

Crystal structure of human indoleamine 2,3-dioxygenase: catalytic mechanism of O2 incorporation by a heme-containing dioxygenase

Human indoleamine 2,3-dioxygenase (IDO) catalyzes the cleavage of the pyrrol ring of L-Trp and incorporates both atoms of a molecule of oxygen (O2). Here we report on the x-ray crystal structure of human IDO, complexed with the ligand inhibitor 4-phenylimidazole and cyanide. The overall structure of IDO shows two alpha-helical domains with the heme between them. A264 of the flexible loop in the heme distal side is in close proximity to the iron. A mutant analysis shows that none of the polar amino acid residues in the distal heme pocket are essential for activity, suggesting that, unlike the heme-containing monooxygenases (i.e., peroxidase and cytochrome P450), no protein group of IDO is essential in dioxygen activation or proton abstraction. These characteristics of the IDO structure provide support for a reaction mechanism involving the abstraction of a proton from the substrate by iron-bound dioxygen. Inactive mutants (F226A, F227A, and R231A) retain substrate-binding affinity, and an electron density map reveals that 2-(N-cyclohexylamino)ethane sulfonic acid is bound to these residues, mimicking the substrate. These findings suggest that strict shape complementarities between the indole ring of the substrate and the protein side chains are required, not for binding, but, rather, to permit the interaction between the substrate and iron-bound dioxygen in the first step of the reaction. This study provides the structural basis for a heme-containing dioxygenase mechanism, a missing piece in our understanding of heme chemistry.     

Hepatic hyperplastic nodules showing stains by portal blood flow: hemodynamics revealed by CTAP and CTHA

The hepatic hyperplastic nodule associated with idiopathic portal hypertension is classified as portal blood flow and hepatic arterial blood flow dominant types. These nodular lesions are considered attributable to abnormal blood flow in the liver. We describe a rare case of hepatic hyperplastic nodules showing stains by portal blood flow.     

Dendritic cells that endocytosed antigen-containing IgG-liposomes elicit effective antitumor immunity

Liposomes represent a promising vehicle to deliver exogenous antigens to dendritic cells (DCs) for tumor immunotherapy. Targeting exogenous antigens to Fcgamma receptors on DCs has been shown to result in efficient presentation of antigen-derived peptides on major histocompatibility complex (MHC) class I and class II molecules. In this study, it was investigated whether DCs that endocytosed physicochemically optimized antigen-containing liposomes conjugated with IgG efficiently present antigens on MHC class I and class II molecules, and consequently induce strong antitumor immune responses. IgG-conjugated liposomes that were 200 nm in diameter without attaching polyethylene glycol were most efficiently endocytosed by DCs. Human monocyte-derived DCs that endocytosed tetanus toxoid (TT)-containing IgG liposomes via CD32 stimulated CD4(+) T cells more strongly than DCs pulsed with TT-containing bare liposomes or with soluble TT. Immunization of mice with DCs that endocytosed ovalbumin (OVA)-containing IgG liposomes but not OVA-containing bare liposomes or soluble OVA completely prevented the growth of OVA-expressing lymphoma cells. Importantly, administration of DCs that endocytosed OVA-containing IgG liposomes to the mice with established OVA-expressing tumors strongly suppressed tumor growth. This study demonstrates an IgG liposome with physicochemical properties suitable for delivering antigens to DCs and paves the way to the application of IgG liposomes for tumor immunotherapy using DCs.     

STI571 (Glivec) induces cell death in the gastrointestinal stromal tumor cell line, GIST-T1, via endoplasmic reticulum stress response

STI571 is a specific inhibitor of tyrosine kinases, such as BCR-ABL, platelet-derived growth factor receptor, and c-KIT, and has recently been approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors (GISTs). This study demonstrated that STI571 induces cell death in the gastrointestinal stromal tumor cell line, GIST-T1. In these cells, STI571 induced pro-caspase-12 or pro-caspase-7 cleavage and it affected caspase-3 activity and induced the endoplasmic reticulum (ER)-resident chaperone, glucose-regulated protein 78. The STI571-induced cell death was blocked by the protein synthesis inhibitor, cycloheximide. Together, these results suggest that STI571 induces cell death in GIST-T1 cells, at least in part, via the ER stress response.     

Preparation of prefabricated vascularized bone graft with neoangiogenesis by combination of autologous tissue and biodegradable materials

Prefabricated vascularized bone grafts have previously been prepared by combining an autologous vessel bundle with auto-particulate cancellous bone and marrow (PCBM) and a biodegradable membrane. Only small quantities of low-density vascularized bone tissue have been formed using this method. The authors of the present study combined beta-tricalcium phosphate (beta-TCP), a biodegradable ceramic, with a prefabricated vascularized bone graft to augment osteogenesis. A saphenous vessel bundle from a rat was wrapped in a biodegradable membrane. Then, autologous PCBM was mixed with beta-TCP granules and packed into the rolled membrane. In the control group, beta-TCP was omitted. Bone formation was histologically assessed 6 and 9 weeks after implantation. The volume of newly formed bone tissue in the rolled membrane was greater in the presence of beta-TCP granules than in the control. Microvessels had formed throughout the new bone tissue. When a prefabricated vascularized bone graft was onlay-grafted to the femur of the same rat, the prefabricated vascularized bone graft directly fused to the cortical surface of the femur, with no intervening fibrous tissue. These findings suggest that beta-TCP in combination with PCBM enhances the volume and density of bone tissue in prefabricated vascularized bone grafts.     

Comparison of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay to pulsed-field gel electrophoresis to determine the effect of repeated subculture and prolonged storage on RFLP patterns of Shiga toxin-producing Escherichia coli O157:H7

In this study, we compared a recently developed PCR-restriction fragment length polymorphism (PCR-RFLP) assay with pulsed-field gel electrophoresis (PFGE) using three different Shiga toxin-producing Escherichia coli (STEC) strains to understand whether repeated subculture in vitro and prolonged storage at room temperature affect the RFLP patterns of STEC. The PFGE profiles of the STEC strains changed by 1 to 8 fragments after repeated subculture and prolonged storage; one strain was no longer clonal after repeated subculture compared to the original isolate according to the Tenover criteria. In contrast, RFLP patterns obtained by PCR-RFLP were identical after repeated subculture and prolonged storage. These data clearly indicate that the PCR-RFLP assay which is based on the diversity of region V, a regulatory region of Stx-phage, was not affected by repeated subculture and prolonged storage and is a more practical and reliable method for molecular typing of STEC strains.     

The lethal toxicity of pancreatic ascites fluid in severe acute necrotizing pancreatitis

BACKGROUND/AIMS: The pathogenesis and the molecular mechanisms of the development and progression of the acute pancreatitis (AP) are not clearly understood. Ascites fluid is known to be important in the clinical progression of AP. We present the lethal toxicity of human pancreatic ascites fluid for experimental pancreatitis, with the therapeutic course of severe necrotizing AP. METHODOLOGY: The material was in a 33-year-old male admitted with epigastric pain. An abdominal CT revealed that his pancreas was swollen and contained pancreatic fluid collection extending to the pelvic cavity. He had complicated acute renal failure, sepsis, and DIC, and received hemodialysis, and continuous arterial infusion therapy (CAI). The peripancreatic infection was acquired, and percutaneous interventional radiology (IVR) was performed for the abscess drainage. The drained liquid around the pancreas contained high molecular cytokines, protease, and bacterial contamination. To evaluate the toxicity of the ascites fluid, we gave it intraperitoneally to rats in which pancreatitis had been induced and rats that had undergone a sham operation; these rats died immediately. The consistent irrigation and drainage of the abscess was administered and the patient's general condition improved. At this time, we gave the drained material intraperitoneally to other rats with induced pancreatitis and sham operation, but all these rats survived. RESULTS: These experimental results suggested that pancreatitis-associated ascites fluid contained a lethal toxicity. For curing this disease, elimination of these potential toxic mediators was essential. Our intensive IVR-based therapy improved the patient's prognosis. CONCLUSIONS: Downregulating this inflammatory process leads to a decrease in the mortality of severe acute pancreatitis.     

Frontal white matter anisotropy and symptom severity of late-life depression: a magnetic resonance diffusion tensor imaging study

OBJECTIVES: To investigate the disruption of neural circuits in the frontal lobes and limbic structures in late-life depressed patients compared with healthy controls, and to examine the correlation between the degree of microstructural abnormalities of white matter and clinical symptom severity in late-life depression. METHODS: Thirteen patients with late-life depression and matched control subjects underwent diffusion tensor imaging. Fractional anisotropy (FA), an index of the integrity of white matter tracts, was determined in the white matter of frontal, temporal, and occipital brain regions and the corpus callosum. RESULTS: A significant reduction was found in white matter FA values of widespread regions of the frontal and temporal lobes of depressed patients. Also, there was some evidence suggesting that white matter FA values of the inferior frontal brain region are inversely related to severity of depression. CONCLUSIONS: These results suggest the possible loss of integrity within frontal and temporal white matter fibre tracts and implicate the orbitofrontal circuit in symptom severity in late-life depression.     

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo. In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A₂ receptor antagonist (5 μM), a stable prostacyclin (0.55 nM), a stable prostaglandin E₁ (141 nM) and a phosphodiesterase inhibitor (100 μM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.