Affinity imaging mass spectrometry (AIMS): high-throughput screening for specific small molecule interactions with frozen tissue sections.

A novel screening system, using affinity imaging mass spectrometry (AIMS), has been developed to identify protein aggregates or organ structures in unfixed human tissue. Frozen tissue sections are positioned on small (millimetre-scale) stainless steel chips and incubated with an extensive library of small molecules. Candidate molecules showing specific affinity for the tissue section are identified by imaging mass spectrometry (IMS). As an example application, we screened over a thousand compounds against Alzheimer's disease (AD) brain tissue and identified several compounds with high affinity for AD brain sections containing tau deposits compared to age-matched controls. It should also be possible to use AIMS to isolate chemical compounds with affinity for tissue structures or components that have been extensively modified by events such as oxidation, phosphorylation, acetylation, aggregation, racemization or truncation, for example, due to aging. It may also be applicable to biomarker screening programs.

HrpZ harpins from different Pseudomonas syringae pathovars differ in molecular interactions and in induction of anion channel responses in Arabidopsis thaliana suspension cells.

HrpZ, a type three secretion system helper protein from the plant-pathogen Pseudomonas syringae, can be recognized by many plants as a defence elicitor. Responses of Arabidopsis thaliana suspension cells to different HrpZ variants were studied by electrophysiological methods and cell death assay. Purified HrpZ originating from a compatible pathogen P. syringae pv. tomato DC3000 (HrpZ(Pto)) and incompatible P. syringae pv. phaseolicola (HrpZ(Pph)) both promoted Arabidopsis cell death. As an early response, both HrpZ variants induced an increase in time dependent K(+) outward rectifying current. In contrast, the effects of HrpZ proteins on anion currents were different: HrpZ(Pph) had no effect, and HrpZ(Pto) induced an anion current increase. This suggests that the observed responses of the K(+) channels and anion channels resulted from different and separable interactions and that the interaction implied in anion current modulation is host-specific. HrpZ(Pto) and HrpZ(Pph) also had a different sequence preference in phage display screen for peptide-binding. These peptides presumably represent a part of a putative target protein in the host, and HrpZ proteins of different P. syringae pathovars might have different binding specificities to match the allelic variation between plant species. Supporting the idea that the peptide-binding region of HrpZ is important for interactions with host cell components, we found that a mutation in that region changed the anion channel response of Arabidopsis cells.

Altered activity of lysophospholipase D, which produces bioactive lysophosphatidic acid and choline, in serum from women with pathological pregnancy.

Altered lipid metabolism is associated with human abnormal pregnancy, such as pre-eclampsia and preterm labor, and potentially leads to fetus loss. A causative factor for the onset and progress of the systemic multifactorial syndromes associated with the pathological pregnancy is oxidized low-density lipoprotein, an active identity of which was postulated to be lysophosphatidic acid (LPA). We previously found that LPA is produced extracellularly by plasma lysophospholipase D (lysoPLD) activity of autotaxin, a tumor cell motility-stimulating protein. In this study, a convenient assay based on the choline released from endogenous substrate or exogenous lysophosphatidylcholine (LPC) was used for comparison of serum lysoPLD activity among patients with normal and abnormal pregnancy. The serum choline-producing activity was found to be mainly due to autotaxin, and dependent on its dilution rate. There was some association between low dilution dependency of serum lysoPLD activity toward an exogenous LPC and high lysoPLD activity toward endogenous substrates in cases of patients with preterm labor and pre-eclampsia. However, there was no difference in the serum level of LPC between women with normal pregnancy and those with pathological pregnancy. These results indicate that production of bioactive LPA by lysoPLD activity is elevated by an unknown mechanism that may be related to increased availability of endogenous substrates LPC, but not its concentration in human serum. If the level of LPA in blood circulation is elevated in the pathological pregnancies in vivo, it may play a role in induction and/or progression of systemic vascular dysfunction seen patients with preterm labor or pre-eclampsia.

How can we increase living related donor renal transplantations?

BACKGROUND: In Japan, living donor renal transplantation has gained momentum due to an increased number of patients with end-stage renal disease. Living donation not only provides better outcomes, but also the recipients usually need less medications, thereby increasing the quality of life and reducing the potential side effects of immunosuppression. MATERIALS AND METHODS: For the past 25 years, our center had performed 140 open donor nephrectomy (OPNx) renal transplantations. Since July 2003, we changed our procurement operation to living hand-assisted laparoscopic donor nephrectomy (HALNx) in 49 cases. Our operative technique consisted of two 12-mm ports placed in the midaxillary line at the superior and inferior levels of the umbilicus. Next, a 5-cm incision was made in the midline periumbilicus and the hand port system fitted through a midline abdominal incision. RESULTS: In 49 cases, HALNx was completed successfully; no patient required conversion to laparotomy. The estimated blood loss was 33.0 +/- 43.4 g and no patient required blood transfusion. In comparison, in OPNx the blood loss was 426.5 +/- 247.6 g (P < .001). The mean operative times were 167.4 +/- 39.7 minutes for HALNx and 228.4 +/- 35.7 minutes for OPNx (P < .001). The postoperative hospital stays were 9.1 +/- 3.8 days for HALNx and 13.0 +/- 1.9 days for OPNx (P < .001). For 3 years prior to introduction of HALNx, we had performed only 10 living donor renal transplantations. Since the introduction of HALNx in 2003, the number of living donors has tripled during the following 3 years. CONCLUSIONS: Herein we have reported that HALNx was superior in terms of less operative time and blood loss, postoperative pain and recovery, and shorter hospital stay. Overall donor patient satisfaction was also better in the HALNx group. HALNx is a safe procedure that makes kidney donation more appealing to potential live donors and has increased the living donor pool at our center.

The role of plasmapheresis therapy for perioperative management in ABO-incompatible adult living donor liver transplantation.

BACKGROUND: Although living donor liver transplantation (LDLT) was established as a treatment for end-stage liver disease in Japan, the indication for LDLT across an ABO-incompatible barrier remains controversial. The purpose of this study was to elucidate the role of plasmapheresis in incompatible LDLT. METHODS: Eleven adult patients (seven men and four women) who underwent incompatible LDLT were enrolled in this study. Of these three patients had hepatocellular carcinoma, three chronic hepatitis C, one Wilson's disease, one autoimmune hepatitis, one chronic hepatitis B, one hemochromatosis, and one fulminant hepatic failure. The immunosuppressive regimen consisted of tacrolimus, prednisolone, mycophenolate mofetil (or cyclophosphamide), and prostaglandin E1 in all patients. Multiple plasmapheresis was performed perioperatively to reduce the recipient's antibody titers against the donor's blood type. RESULTS: Plasmapheresis was useful for the reduction of the recipient's antibody titers to x 16 or lower before and after transplantation. There was no difference in transplant outcome between the 11 patients with incompatible blood group and 30 patients with identical or compatible blood groups. DISCUSSION: Major postoperative complications such as intrahepatic biliary complications and hepatic necrosis may occur in incompatible transplantation. Several investigators suggested that anti-immunoglobulin (Ig) M and anti-IgG antibody titers sustained these complications. The antibody titers must be decreased sufficiently with plasmapheresis. An elevation of anti-ABO titers after transplantation may be a predictive risk factor for increased mortality and morbidity. In order to perform LDLT in a safer manner, plasmapheresis is an indispensable treatment to improve the outcome of ABO-incompatible cases.

Successful case of adult ABO-incompatible liver transplantation: beneficial effects of intrahepatic artery infusion therapy: a case report.

BACKGROUND: In Japan ABO-incompatible liver transplantation has been done on >100 occasions up to 2003. However, <30% are cases involving adults. The difficultly of ABO-incompatible liver transplantation is associated with the high frequency of humoral rejection and local disseminated intravascular coagulation (DIC), leading to many postoperative complications. We report a successful case of adult ABO-incompatible liver transplantation with the use of an intrahepatic artery infusion. METHODS: A 36-year-old man with Wilson disease, underwent living donor liver transplantation from an ABO-incompatible donor. The immunosuppressive therapy included multiple perioperative plasmaphereses, splenectomy, and treatment with tacrolimus, methylprednisolone, and cyclophosphamide. The dose and blood level of tacrolimus were the same as in ABO-compatible cases. In addition to these therapies, we administered an intrahepatic arterial infusion with prostaglandin (PG) E1 alone. RESULTS: After perioperative plasmapheresis and cyclophosphamide, antidonor blood group antibody titers remained undiluted and without vascular complications throughout the postoperative course, but there was a tendency for bleeding that continued for 10 days after transplantation. On postoperative day 10, a reexploration was performed for intraabdominal bleeding. During another operation on postoperative day 59 a biloma was found and drained. The patient has now survived for 120 days after transplantation with normal liver function. CONCLUSIONS: Beneficial effect of intrahepatic artery infusion with PGE1 seems to be useful in adult ABO-incompatible liver transplantation.

Acute brain injury in hypoglycaemia-induced hemiplegia.

BACKGROUND: The development of hemiplegia as a result of hypoglycaemia was first described in 1928. However, the mechanism remains unclear. CASE REPORT: We report a case of a 58-year-old male with diabetes, who developed left hemiplegia during a severe hypoglycaemic event. Results Diffusion-weighted magnetic resonance imaging detected an increased signal intensity in the pons, indicating that the patient's hemiplegia resulted from acute brain injury. CONCLUSIONS: This report provides evidence that acute brain injury may be a cause of the neurological deficit.

mRNA stability and the secretion signal of HrpA, a pilin secreted by the type III system in Pseudomonas syringae.

Gram-negative bacteria that are pathogenic for animals or plants utilise a specialised Type III secretion system to inject effector proteins into their eukaryotic target cells. The basis for selection of the proteins to be translocated via type III systems is still enigmatic. No clearly defined consensus amino acid sequence that could serve as a specific secretion signal has been identified, and the hypothesis that an mRNA secondary structure acts as the signal has several shortcomings. We have localised a secretion signal that is sufficient to ensure the secretion of the pilin HrpA, a substrate and an indispensable extracellular component of the type III secretion machinery of Pseudomonas syringae pv. tomato DC3000, to the first 15 codons. Transcription of hrpA starts at a single site 42 bp upstream of the first codon. Gene swapping experiments revealed that altering the continuity of the 5' non-translated leader with the region including the secretion signal radically decreased accumulation of the hrpA transcript. These results indicate that an mRNA secondary structure, possibly formed in this region, is important for efficient expression of the gene. The proposed secondary structure is not, however, indispensable for the secretion of HrpA and it does not couple secretion and translation.

Early patterning of the prospective midbrain-hindbrain boundary by the HES-related gene XHR1 in Xenopus embryos.

The molecular mechanisms that govern early patterning of anterior neuroectoderm (ANE) for the prospective brain region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1). XHR1 is specifically expressed in the midbrain-hindbrain boundary (MHB) region at the tailbud stage. The localized expression of XHR1 was detected as early as the early gastrula stage in the presumptive MHB region, an area just anterior to the involuting dorsal mesoderm that is demarcated by the expression of the gene Xbra. Expression of XHR1 was detected much earlier than that of other known MHB genes, XPax-2 and En-2, and also before the formation of the expression boundary between Xotx2 and Xgbx-2, suggesting that the early patterning of the presumptive MHB is independent of Xotx2 and Xgbx-2. Instead, the location of XHR1 expression appears to be determined in relation to the Xbra expression domain, since reduced or ectopic expression of Xbra altered the XHR1 expression domain according to the location of Xbra expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1 in the MHB region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1. Furthermore, ectopically expressed wild-type XHR1 near the MHB region enhanced En-2 expression only in the MHB region but not in the region outside the MHB. These data suggest that XHR1 is required, but not sufficient by itself, to initiate MHB marker gene expression. Based on these data, we propose that XHR1 demarcates the prospective MHB region in the neuroectoderm in Xenopus early gastrulae.

The relationship of primary tumor thickness in carcinoma of the tongue to subsequent lymph node metastasis.

OBJECTIVES: To study whether primary tumor thickness of stage I/II tongue carcinoma provides information about subsequent lymph node metastasis. METHODS: Twenty consecutive patients with T1N0M0 or T2N0M0 tongue carcinoma were studied. Primary tumor thickness was measured with post-contrast helical computed tomography or intra-oral sonography. Cervical lymph nodes were evaluated periodically with sonography at intervals of 2-4 weeks. Sensitivity, specificity and accuracy for subsequent metastasis was calculated. RESULTS: Positive sonographic findings appeared in nine nodes of nine patients during this follow-up period. Eleven patients underwent neck dissections, and nine had histopathologically positive nodes. Nine patients had no sonographic findings of metastasis during a minimum follow-up period of 20 months. Primary tumor thickness varied from 3-16 mm. Using 5 mm as a cut-off thickness, the sensitivity, specificity and accuracy for subsequent lymph node metastasis were 64, 100 and 75% respectively. CONCLUSIONS: Patients with stage I/II tongue carcinoma which is more than 5 mm thick are more likely to develop lymph node metastasis.

Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster.

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.

Successful living donor liver transplantation for polycystic liver in a patient with autosomal-dominant polycystic kidney disease.

Orthotopic liver transplantation has been recommended for patients with disabling polycystic liver disease (PCLD). Because of the shortage of cadaveric donors, living donor liver transplantation (LDLT) has been developed as an alternative. We describe the case of a woman with PCLD as an extrarenal manifestation of autosomal-dominant polycystic kidney disease (ADPKD) who was successfully palliated by LDLT. The patient was a 48-year-old woman with abdominal distention. Computed tomography showed a massively enlarged liver containing innumerable cysts, as well as bilateral kidney cysts. Hepatic and renal functions were well preserved. Genetic analysis of the family did not exclude linkage to the PKD1 locus. Two and a half years after the first examination, the patient reported severely disabling symptoms caused by the PCLD. Living donor liver transplantation was performed using a right-lobe graft. The recipient and donor were both well 8 months after the transplantation. The excised liver weighed 7.4 kg, and the histopathology revealed multiple cysts and von Meyenburg complexes in the portal areas.

Cholangiocarcinoma with a background of hepatitis B virus-associated cirrhosis.

Recently, hepatitis virus-associated chronic hepatitis or cirrhosis has been suggested to be involved in the pathogenesis of cholangiocarcinoma (CC). A 52-year-old man was diagnosed as CC with a background of hepatitis B virus (HBV)-dependent cirrhosis. A minute hepatic tumor was found during the follow-up, and was diagnosed as CC on percutaneous biopsy. The patient died of hepatic failure and an autopsy revealed the tumor to be a well to moderately differentiated adenocarcinoma. An immunohistological analysis of HBV X gene-encoded protein (HBX) was neither detected in the cancerous nor in the noncancerous tissue. No oncogenic role of the virus was verified in this case.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Changes in isoprenoid lipid synthesis by gemfibrozil and clofibric acid in rat hepatocytes.

We studied whether gemfibrozil and clofibric acid alter isoprenoid lipid synthesis in rat hepatocytes. After incubation of the cells with the agent for 74 hr, [(14)C]acetate or [(3)H]mevalonate was added, and the cells were further incubated for 4 hr. Gemfibrozil and clofibric acid increased ubiquinone synthesis from [(14)C]acetate and [(3)H]mevalonate. The effect of gemfibrozil was greater than that of clofibric acid. Also, gemfibrozil decreased dolichol synthesis from [(14)C]acetate and [(3)H]mevalonate. However, clofibric acid increased dolichol synthesis from [(3)H]mevalonate. Gemfibrozil decreased cholesterol synthesis from [(14)C]acetate and [(3)H]mevalonate. Clofibric acid decreased cholesterol synthesis from [(14)C]acetate, but did not affect synthesis from [(3)H]mevalonate. These results suggest that both agents, at different rates, activate the synthetic pathway of ubiquinone, at least from mevalonate. Gemfibrozil may inhibit the synthetic pathway of dolichol, at least from mevalonate. Contrary to gemfibrozil, clofibric acid may activate the synthetic pathway of dolichol from mevalonate. Gemfibrozil may inhibit the synthetic pathway of cholesterol from mevalonate in addition to the pathway from acetate to mevalonate inhibited by both agents.

Opsonic function and concentration of human serum ficolin/P35.

Collectins, C-type (Ca2+-dependent) animal lectins with both collagenous and carbohydrate recognition domains, function as opsonins against pathogens. We previously described an N-acetylglucosamine (GlcNAc)-binding lectin (ficolin/P35) with a collagen- and a fibrinogen-like sequence present in human serum. In this report we show that ficolin/P35 can serve as an opsonin and enhance the clearance of pathogens having surface GlcNAc. Ficolin/P35 bound to an Ra chemotype strain of Salmonella typhimurium (TV119) which has an exposed GlcNAc at the non-reducing termini of the polysaccharide. On the other hand, ficolin/P35 did not bind to LT2, a smooth type strain of S. typhimurium with additional O-polysaccharides covering GlcNAc. Ficolin/P35 enhanced the uptake of TV119 by monocytes or polymorphonuclear leukocytes but had no opsonic activity towards LT2. These results suggest that, like collectins, ficolin/P35 is a collagenous lectin which has a role in innate immunity against certain pathogenic organisms by acting as an opsonin. We prepared monoclonal antibodies against ficolin/P35 and developed an enzyme-linked immunosorbent assay (ELISA) for measuring ficolin/P35 concentrations in humans. The mean serum concentration of ficolin/P35 from 130 normal individuals was estimated to be 13.7 microg/ml.

Mutational analysis of the Pseudomonas syringae pv. tomato hrpA gene encoding Hrp pilus subunit.

Plant pathogenic Pseudomonas syringae strains harbour a type III secretion pathway suggested to be involved in the delivery of effector proteins from the bacteria into plant cells. During plant interaction, the bacteria apparently produce surface appendages, termed Hrp pili, that are indispensable for the secretion process. We have created an insertion mutation library, as well as deletion mutations to hrpA, the structural gene encoding Hrp pilin. Analysis of the mutants revealed gene regions important for hrpA expression, pilus assembly and pilus-dependent autoagglutination of the bacteria. The majority of insertions in the amino-terminal half of the pilin were tolerated without bacterial interaction with plants being affected, while the carboxy-terminus appeared to be needed for pilus assembly. Insertions in the 5' non-translated region and the first codons within the open reading frame affected mRNA production or stability and abolished protein production.

Intracortical hemangioma of the mandible. Case report.

We describe the first reported case of an intracortical hemangioma of the mandible in a 13-year-old Japanese girl. Panoramic radiography and CT demonstrated a small osteolytic lesion which had expanded and thinned the cortex at the inferior border of the left mandible. The lesion enhanced on post-contrast T1-weighted MRI. The diagnosis was confirmed by histopathology following block excision. Intracortical hemangioma should be considered in the differential diagnosis of radiolucent lesions orf the mandible