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Objective: To investigate the superiority in 2 radical prostatectomies, we compared the initial results of robotic-assisted radical prostatectomy (RARP) to those of retropubic radical prostatectomy (RRP) performed during the same period at Nagakubo hospital., Patients and Methods: The study was conducted on a total of 160 patients having undergone radical prostatectomy from April 2009 to March 2012 (92 patients with RARP and 68 with RRP). We investigated surgical stress, cancer control, functional outcomes and complications in both groups., Results: Surgical stress; operation time was significantly shorter with RRP; however, blood loss and serum total protein loss were significantly less with RARP. White blood cell count at 2 days after surgery was significantly less with RARP. The rates of analgesic use and SIRS were similar. Although the date on which taking solid meals resumed did not differ, the duration of indwelling urethral catheter and admission period were significantly shorter with RARP. Cancer control; the rates of positive surgical margin were 27.2% and 19.1% with RARP and RRP, respectively (p = 0.24), and biochemical recurrence was seen in 12.0% and 19.1% with RARP and RRP, respectively (p = 0.73), which were not significantly different. Continence; urinary continence outcomes with RARP and RRP were 17% and 4% for urinary continence at discharge (p = 0.01), 1.8 and 3.3 months for no more than one pad per day (p < 0.01), and 4.3 and 6.2 months for pad free (p = 0.03), respectively. Sexual function; erection recovery within 6 mo was only observed with RARP; however, overall recovery rate of erection was 65% and 75% with RARP and RRP, respectively (p = 0.69)., Complications: 1 case with a rectal injury was seen in both groups, but complication rates were 8.7% and 16.2% with RARP and RRP, respectively (p = 0.22)., Conclusion: In spite of our initial experience of RARP, surgical stress and complications with RARP were considered to be superior to that with RRP. Cancer control and sexual function showed no significant difference between RARP and RRP, however, urinary continence outcome is significantly superior with RARP. Our data suggest that treatment outcome after initial experience with RARP is not inferior to that with RRP, and better results are expected by improving surgical techniques.

Mucosa-associated lymphoid tissue (MALT) lymphoma occurs in various sites, but rarely in the urinary tract. Imaging studies of a 69-year-old male revealed a left hydronephrosis and diffuse thickening of the renal pelvic and upper ureteral wall. Retrograde pyelography revealed a narrowing in this region, and brush cytology specimens contained atypical cells. As the lesion was considered to be malignant, surgical excision was performed. Histological analysis revealed an intense lymphoid infiltrate mainly consisting of B cells. The immunohistochemistry results demonstrated that the lesion was positive for CD20 and CD79a and negative for CD5 and cyclin D1. These findings led to a diagnosis of MALT lymphoma. Pathological exploration disclosed a focally dense invasion of lymphoma cells into not only the renal pelvis, but the whole ureteral wall and surrounding tissue; therefore, the patient underwent eight courses of rituximab treatment. Diffuse invasion of lymphoma cells into the whole ureter was rarely observed. Since the surgery, the patient has survived for 78 months without evidence of a recurrence of lymphoma.

A new simian-human immunodeficiency virus (SHIV), carrying env from an uncloned HIV-1 subtype C clinical isolate (97ZA012), was generated through intracellular homologous recombination, a DNA repair mechanism of the host cell. PCR fragments amplified from an existing SHIV plasmid (a 7-kb fragment from the 5' end and a 1.5-kb fragment from the 3' end) and a 4-kb fragment amplified from 97ZA012 cDNA containing env were co-transfected to human lymphoid cells. The resulting recombinant was subjected to serial passage in rhesus peripheral blood mononuclear cells (RhPBMCs). The resulting SHIV 97ZA012 was replication competent in RhPBMCs and monkey alveolar macrophages, and possessed CCR5 preference as an entry co-receptor. Experimental infection of rhesus macaques with SHIV 97ZA012 caused high titers of plasma viremia and a transient but profound depletion of CD4(+) T lymphocytes in the lung. Animal-to-animal passage was shown to be a promising measure for further adaptation of the virus in monkeys., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: To investigate the safety and efficacy of mirabegron for patients with overactive bladder (OAB) that is unresponsive to antimuscarinic agents or is related to benign prostatic hyperplasia (BPH)., Methods: Fifty-two newly diagnosed OAB patients (M group) and 45 patients with OAB that was unresponsive to antimuscarinics (S group) received mirabegron 50 mg once daily and were evaluated by OAB symptom score (OABSS), IPSS-QOL index, and IPSS at the time of baseline, 4 and 8 weeks. Newly diagnosed OAB patients treated with antimuscarinic agents were compared as controls., Results: Mirabegron was effective for 85.2 % in M group. Significant improvements were seen in each domain of OABSS, and there was no significant difference with antimuscarinic therapy. Mirabegron was efficacious for 61.6 % of S group, and significant decreases of OABSS and IPSS-QOL index were observed. Significant improvements were also seen in voiding symptoms in men. Post-void residual urine volumes before and after treatment were 32.1 and 34.8 ml, and 26.2 and 31.3 ml in M and S group, respectively, and there was no significant difference. The incidence of adverse events was 8.4 %, although none were serious, and the patients recovered spontaneously after mirabegron was discontinued., Conclusion: The present study suggests mirabegron is as effective as antimuscarinics for OAB. It improves OAB symptoms in patients with OAB for which antimuscarinic agents are insufficient. This study revealed that mirabegron improves not only OAB symptoms related to BPH, but also voiding symptoms in men. Low and mild incidences of side effects support the safe utility of mirabegron.

Phosphorus supply is a major factor responsible for reduced crop yields. As a result, plants utilize various adaptive mechanisms against phosphorus depletion, including lipid remodelling. Here we report the involvement of a novel plant lipid, glucuronosyldiacylglycerol, against phosphorus depletion. Lipidomic analysis of Arabidopsis plants cultured in phosphorus-depleted conditions revealed inducible accumulation of glucuronosyldiacylglycerol. Investigation using a series of sulfolipid sulfoquinovosyldiacylglycerol synthesis-deficient mutants of Arabidopsis determined that the biosynthesis of glucuronosyldiacylglycerol shares the pathway of sulfoquinovosyldiacylglycerol synthesis in chloroplasts. Under phosphorus-depleted conditions, the Arabidopsis sqd2 mutant, which does not accumulate either sulfoquinovosyldiacylglycerol or glucuronosyldiacylglycerol, was the most severely damaged of three sulfoquinovosyldiacylglycerol-deficient mutants. As glucuronosyldiacylglycerol is still present in the other two mutants, this result indicates that glucuronosyldiacylglycerol has a role in the protection of plants against phosphorus limitation stress. Glucuronosyldiacylglycerol was also found in rice, and its concentration increased significantly following phosphorus limitation, suggesting a shared physiological significance of this novel lipid against phosphorus depletion in plants.

Aims: We report the case of a 58-year-old female patient who consulted our Department complaining of positional vertigo and showing spontaneous upbeat nystagmus (UBN) in darkness., Method: We analyzed her UBN three-dimensionally. The MRI scan revealed the astrocytoma in the left cerebellum involving the cerebellar vermis., Result: Three-dimensional analysis showed a spontaneous UBN rotating around the intra-aural axis in the pitch plane., Conclusion: Since the cerebellar vermis is known to plays an inhibitory role on the central vertical vestibule-ocular reflex (VOR), the present results suggest that the spontaneous UBN in darkness observed in this patient was induced by an imbalance of central vertical VOR tone., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: To investigate good indications of photoselective vaporization of the prostate (PVP) by retrospective evaluation of its efficacy in relation to prostate volume., Patients and Methods: A cohort of 400 patients had International Prostate Symptoms Score (IPSS), Quality of Life (QoL) score, urinary peak flow (Q(max) ), and prostate volume (P(vol) ), assessed before and after surgery. Changes of outcome variables were compared among four groups classified according to preoperative prostate volume: <30 mL (group A), ≥30 and <50 mL (group B), ≥50 and <70 mL (group C), and >70 mL (group D)., Results: Operating times and amounts of energy applied were 44.2 min and 159 kJ, 67.7 min and 268 kJ, 111 min and 409 kJ, and 171 min and 604 kJ in groups A, B, C, and D, respectively. All variables improved significantly and immediately, and remained for more than 12 months. Investigation of groups classified by prostate size demonstrated that IPSS and QoL score decreased and improvement rate of Q(max) rose as prostate volume increased. Changes of P(vol) were from 24.5 mL to 13.2 mL (-46.1%), from 39.8 mL to 22.9 mL (-42.4%), from 58.4 mL to 32.8 mL (-43.8%), and from 92.1 mL to 53.8 mL (-41.5%) in groups A, B, C, and D, respectively. Overall the P(vol) reduction rate was 43.2%, and no significant differences were observed in reduction ratios among the groups. Complications were minimal, but bladder neck contracture arose in 16 cases with a smaller prostate., Conclusion: Whilst the percentage reduction in volume was similar across the groups, the larger absolute volume reduction in patients with larger prostates was associated with greater improvement in objective urinary function variables, and with symptom score reduction., (© 2011 THE AUTHORS. BJU INTERNATIONAL © 2011 BJU INTERNATIONAL.)

Objective: To assess the efficacy and safety of transurethral resection in saline vaporization for symptomatic bladder outlet obstruction and the change in prostate volume., Methods: A total of 106 patients with symptomatic bladder outlet obstruction underwent transurethral resection in saline vaporization. The effectiveness of the treatment was evaluated using the International Prostate Symptom Score (IPSS), quality of life score, urinary peak flow, and prostate volume preoperatively and at 1, 3, 6, and 12 months postoperatively. To assess the treatment effect, the patients were divided into 3 groups according to the preoperative prostate volume: group 1 (<45 cm(3), n = 40), group 2 (≥ 45 but <65 cm(3), n = 34), and group 3 (≥ 65 cm(3), n = 32)., Results: The mean perioperative hemoglobin loss was 1.2 g/dL. The mean IPSS, quality of life score, and prostate volume decreased significantly from 24.3, 5.2, and 52.8 cm(3) to 11.1, 2.7, and 29.9 cm(3), respectively (P < .01) and the mean urinary peak flow had increased significantly from 7.3 to 15.1 (P < .01) at 1 month. Moreover, the IPSS, quality of life score, and prostate volume had significantly decreased to 8.8, 2.3, and 26.1 cm(3) at 3 months (P < .05). The investigation of groups classified by prostate size demonstrated that the IPSS in group 3 continued at a significantly lower level than that in groups 1 and 2. The prostate volume decreased gradually and resulted in 52.8% volume reduction for ≤ 12 months. No significant complications were seen; however, irritative symptoms occurred frequently (11.3%)., Conclusion: Transurethral resection in saline vaporization was a safe and effective treatment option and was more efficacious for patients with a larger prostate. The bother scores and prostate volume gradually decreased for ≤ 12 months., (Copyright © 2012 Elsevier Inc. All rights reserved.)

T helper (Th)2 polarized immune responses are characteristically dominant in helminth infections. The gene expression of interferon (IFN)-γ-inducible protein 10 (IP-10/CXCL10), which promotes Th1 responses, in mouse macrophages stimulated with lipopolysaccharide (LPS) and/or IFN-γ was suppressed by excretory/secretory (ES) products of Spirometra erinaceieuropaei plerocercoids. ES products suppressed LPS- and/or IFN-γ-induced transcriptional activities of a luciferase reporter gene under the control of a 243-bp fragment of the IP-10 gene promoter/enhancer, which contains an IFN-stimulated response element (ISRE) and two κB elements. Consistent with this result, ES products inhibited ISRE-dependent heterologous promoter activities and LPS- or IFN-γ-induced ISRE-binding activity. ES products also suppressed LPS-induced IFN-β gene expression. Furthermore, ES products suppressed nuclear factor (NF)-κB RelA (p65)-dependent transcriptional activity, whereas ES products had no effect on the κB-binding activity. These results suggest that ES products suppress the IP-10 gene expression by inhibiting the ISRE- and RelA-dependent transcriptional activities in mouse macrophages., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The malaria transmission-blocking vaccine (TBV) aims to interfere the development of malaria parasite in the mosquito and prevent further transmission in the community. So far only two TBV candidates have been identified in Plasmodium vivax; ookinete surface proteins Pvs25 and Pvs28. The pvs230 (PVX&#95;003905) is reported as an ortholog of Pfs230, a gametocyte/gamete stage TBV candidate in Plasmodium falciparum, however its candidacy for TBV has never been tested. Therefore here, we have investigated whether Pvs230 can be a TBV candidate using P. vivax samples obtained from Thailand. The mouse antiserum raised against the plasmid expressing CRDs I-IV of Pvs230 detected Pvs230 protein in the lysate of P. vivax gametocyte in western blot analysis under non-reducing condition. From the localization of Pvs230 on the outer most regions of gametocyte in the immunofluorescence assay, it appears that Pvs230 is localized on the surface of gametes. Importantly, the anti-Pvs230 mouse serum significantly reduced the number of P. vivax oocysts developed in the mosquito midgut. Moreover, the polymorphism in Pvs230 CRDs I-IV is limited suggesting that it may not be an impediment for the utilization of Pvs230 as an effective TBV candidate. In conclusion, our results show that Pvs230 is a transmission-blocking vaccine candidate of P. vivax., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08&#95;0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum.

Because of ever-increasing environmental deterioration it is likely that the influx of UV-B radiation (280-320 nm) will increase as a result of the depletion of stratospheric ozone. Given this fact it is essential that we better understand both the rapid and the adaptive responses of plants to UV-B stress. Here, we compare the metabolic responses of wild-type Arabidopsis with that of mutants impaired in flavonoid (transparent testa 4, tt4; transparent testa 5, tt5) or sinapoyl-malate (sinapoylglucose accumulator 1, sng1) biosynthesis, exposed to a short 24-h or a longer 96-h exposure to this photo-oxidative stress. In control experiments we subjected the genotypes to long-day conditions as well as to 24- and 96-h treatments of continuous light. Following these treatments we evaluated the dynamic response of metabolites including flavonoids, sinapoyl-malate precursors and ascorbate, which are well known to play a role in cellular protection from UV-B stress, as well as a broader range of primary metabolites, in an attempt to more fully comprehend the metabolic shift following the cellular perception of this stress. Our data reveals that short-term responses occur only at the level of primary metabolites, suggesting that these effectively prime the cell to facilitate the later production of UV-B-absorbing secondary metabolites. The combined results of these studies together with transcript profiles using samples irradiated by 24-h UV-B light are discussed in the context of current models concerning the metabolic response of plants to the stress imposed by excessive UV-B irradiation., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The proteins in apical organelles of Plasmodium falciparum merozoite play an important role in invasion into erythrocytes. Several rhoptry neck (RON) proteins have been identified in rhoptry proteome of the closely-related apicomplexan parasite, Toxoplasma gondii. Recently, three of P. falciparum proteins orthologous to TgRON proteins, PfRON2, 4 and 5, were found to be located in the rhoptry neck and interact with the micronemal protein apical membrane antigen 1 (PfAMA1) to form a moving junction complex that helps the invasion of merozoite into erythrocyte. However, the other P. falciparum RON proteins have yet to be characterized. Here, we determined that "PFL2505c" (hereafter referred to as pfron3) is the ortholog of the tgron3 in P. falciparum and characterized its protein expression profile, subcellular localization, and complex formation. Protein expression analysis revealed that PfRON3 was expressed primarily in late schizont stage parasites. Immunofluorescence microscopy (IFA) showed that PfRON3 localizes in the apical region of P. falciparum merozoites. Results from immunoelectron microscopy, along with IFA, clarified that PfRON3 localizes in the rhoptry body and not in the rhoptry neck. Even after erythrocyte invasion, PfRON3 was still detectable at the parasite ring stage in the parasitophorous vacuole. Moreover, co-immunoprecipitation studies indicated that PfRON3 interacts with PfRON2 and PfRON4, but not with PfAMA1. These results suggest that PfRON3 partakes in the novel PfRON complex formation (PfRON2, 3, and 4), but not in the moving junction complex (PfRON2, 4, 5, and PfAMA1). The novel PfRON complex, as well as the moving junction complex, might play a fundamental role in erythrocyte invasion by merozoite stage parasites., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

A 75-year-old woman was referred to our department for evaluation of a left renal tumor. Computed tomography and other imaging studies demonstrated a left renal mass and tumor extension into the left renal vein passing caudally behind the aorta. We clinically diagnosed the tumor as renal cell carcinoma (RCC) associated with a retroaortic left renal vein thrombus, and performed a radical nephrectomy. Pathological examination of the surgical specimen showed a grade 2, clear cell carcinoma with a renal vein thrombus and negative surgical margin. Retroaortic left renal vein is a rare anomaly with a prevalence of 1.8-2.4%. RCC associated with a retroaortic left renal vein thrombus is rarer still. To our knowledge, this is only the third case report to describe an RCC associated with a tumor thrombus in the retroaortic left renal vein.

A 55-year-old man who had been undergoing hemodialysis for 9 years visited our institution after the sudden onset of severe left flank pain. He presented with hypotension and was admitted immediately because computed tomography (CT) revealed a massive perirenal hematoma. Renal arteriography showed contrast media leakage at the lower branch of the left renal artery, and spontaneous renal rupture was diagnosed. Five months after the bleeding was stopped by selective transcatheter embolization of the branch of renal artery, CT showed an enhanced mass at the upper pole of left kidney and renal cell carcinoma (RCC) was suspected. Radical nephrectomy was performed, the pathological diagnosis was clear cell carcinoma, and the man has not experienced recurrence within 36 months after the surgery. RCC did not appear to be the cause of the original hemorrhage because there was a small residual hematoma in the middle of the renal parenchyma that was separated from the RCC. In cases of spontaneous renal rupture, re-evaluation by imaging studies is mandatory after disappearance of perirenal hematoma because imaging studies at the time of the rupture sometimes do not reveal the cause of the hemorrhage.

Methionine (Met) is an essential amino acid for all organisms. In plants, Met also functions as a precursor of plant hormones, polyamines, and defense metabolites. The regulatory mechanism of Met biosynthesis is highly complex and, despite its great importance, remains unclear. To investigate how accumulation of Met influences metabolism as a whole in Arabidopsis, three methionine over-accumulation (mto) mutants were examined using a gas chromatography-mass spectrometry-based metabolomics approach. Multivariate statistical analyses of the three mto mutants (mto1, mto2, and mto3) revealed distinct metabolomic phenotypes. Orthogonal projection to latent structures-discriminant analysis highlighted discriminative metabolites contributing to the separation of each mutant and the corresponding control samples. Though Met accumulation in mto1 had no dramatic effect on other metabolic pathways except for the aspartate family, metabolite profiles of mto2 and mto3 indicated that several extensive pathways were affected in addition to over-accumulation of Met. The pronounced changes in metabolic pathways in both mto2 and mto3 were associated with polyamines. The findings suggest that our metabolomics approach not only can reveal the impact of Met over-accumulation on metabolism, but also may provide clues to identify crucial pathways for regulation of metabolism in plants.

We report the case of a 28-year-old man who presented with the sole complaint of lumbago. Spinal magnetic resonance imaging (MRI) revealed a solitary, well-defined intramedullary mass at the L1-L2 level typical of a primary spinal cord germinoma. However, cranial magnetic resonance imaging (MRI) showed a concomitant lesion in the suprasellar region. This article describes a rare case of simultaneously detected intracranial and intramedullary spinal cord germinoma and its possible etiopathology.

Malaria kills more than a million people a year, causes malady in about three hundred million people and poses risk to approximately 40% of the world's population living in malarious countries. This disease is re-emerging mainly due to the development of drug-resistant parasites and insecticide-resistant mosquitoes. Therefore, we are now forced to resort to remedy through vaccination. Until now, not even a single licensed malaria vaccine has been developed despite intensive efforts. Even the efficacy of RTS,S, the most advanced and promising vaccine candidate in the pipeline of malaria vaccine development, was only around 50% based on a number of clinical trials. These facts urge malaria researchers to urgently enrich this pipeline, as much as possible, with potential vaccine candidates. With the availability of malaria genome database, the enrichment of this pipeline is possible if we could now employ an efficient protein expression technology to decode the malaria genomic data, without any codon optimization, into quality recombinant proteins. Then, these synthesized recombinant proteins can be characterized and screened for discovering novel potential vaccine targets. The wheat germ cell-free protein synthesis system will be a promising tool to this end. This review highlights the recent successes in synthesizing quality malaria proteins using this tool., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

A 62-year-old woman presented with right flank pain, and computed tomography (CT) showed bilateral adrenal tumors (right, 8 cm; left, 4 cm). There were no abnormal findings on physical examination, and adrenal hormone levels in serum and urine were within normal limits. Radiological studies showed a right adrenal tumor with intratumoral hemorrhage, a cystic tumor in the left adrenal gland, and no sign of distant metastasis. Because laboratory tests showed normal levels of adrenal hormones, we considered the tumors to be nonfunctioning adrenal tumors. The right adrenal tumor was surgically removed because it was thought to be malignant, and histological examination revealed that it was a leiomyosarcoma. Postoperative CT showed a pleural metastasis in the right chest wall. The patient received combination chemotherapy with cyclophosphamide, vincristine, adriamycin, and dacarbazine. The metastasis was also treated with radiofrequency ablation (RFA). Because the left adrenal tumor grew rapidly despite two courses of the chemotherapy, it too was surgically removed. After the operation, metastasis in the right iliac bone was treated with RFA and radiotherapy. Positron emission tomography detected bilateral femoral metastases, and these were treated with radiotherapy in combination with a low dose of cisplatin. A liver metastasis and a small metastasis in the left kidney were treated with RFA and a metastasis in the pancreatic tail was removed surgically. Sixteen months after the right adrenalectomy, the patient died due to systemic spread of the disease. Multiple local treatments including RFA, radiotherapy, and resection were effective for the local control of metastatic lesions of adrenal leiomyosarcoma.

Chronic kidney disease (CKD) is associated with increased risk of cardiovascular disease (CVD), and may also lead to an increase in medical expenditures. The effect of CKD on individual and population medical expenditures was examined in a Japanese cohort study. The participants included 4026 Japanese National Health Insurance beneficiaries aged 40-74 years living in one city, who had no history of major CVD. They were classified into three categories according to their glomerular filtration rate (GFR) at the baseline survey, and their total medical expenditures were followed-up for 3 years. The arithmetic and geometric means of the cumulative medical expenditures were calculated for each category. The geometric mean was also expressed after adjusting for several confounding factors using analysis of covariance. The CKD-related medical expenditures attributable to the participants with mild (60 < or = GFR < 90) or moderate (30 < or = GFR < 60) kidney dysfunction were compared with those of participants with normal (GFR > or = 90) kidney function. There was a negative correlation between the GFR category and the arithmetic and geometric means of personal medical expenditures (adjusted geometric mean: GFR > or = 90, 167,879 yen; 60 < or = GFR < 90, 210,660 yen; and 30 < or = GFR < 60, 330,050 yen). The CKD-related medical expenditures accounted for 11.5 and 6.5% of the total medical expenditures for the participants with mild and moderate kidney dysfunction, respectively. The prevention of mild CKD is very important for containing medical expenditures. Appropriate strategies, such as regular creatinine measurement, are needed for early detection of CKD.

During the last decade transcriptome analyses demonstrated that alternative splicing plays an important role to generate a large number of mRNA and protein isoforms from a limited number of genes. However, the frequency of the alternative splicing dramatically varies among living organisms. For example, 35-65% of human genes are involved in alternative splicing, whereas only a few are reported for unicellular organism yeast. Alternative splicing has been observed for several genes in the deadliest malaria parasite Plasmodium falciparum, but the frequency and the type were not systematically analyzed so far. In this study, we determined partial cDNA sequences for 88 open reading frames surrounding 246 introns in P. falciparum which were transcribed at schizont and gametocyte stages, and observed 15 instances of alternative splicing within a total of 14 gene transcripts, 16% of the analyzed genes. Among 5 basic splicing patterns, alternative 5' and 3' splicing, and intron retention were detected. Alternative splicing in 7 open reading frames had effects on the domain architectures of the gene products, which might result in modifying the cellular localization and function of these products.

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host-pathogen interactions and malaria virulence.

Erythrocyte invasion is an essential step in the establishment of host infection by malaria parasites, and is a major target of intervention strategies that attempt to control the disease. Recent proteome analysis of the closely-related apicomplexan parasite, Toxoplasma gondii, revealed a panel of novel proteins (RONs) located at the neck portion of the rhoptries. Three of these proteins, RON2, RON4, and RON5 have been shown to form a complex with the microneme protein Apical Membrane Protein 1 (AMA1). This complex, termed the Moving Junction complex, localizes at the interface of the parasite and the host cell during the invasion process. Here we characterized a RON2 ortholog in Plasmodium falciparum. PfRON2 transcription peaked at the mature schizont stage and was expressed at the neck portion of the rhoptry in the merozoite. Co-immunoprecipitation of PfRON2, PfRON4 and PfAMA1 indicated that the complex formation is conserved between T. gondii and P. falciparum, suggesting that co-operative function of the rhoptry and microneme proteins is a common mechanism in apicomplexan parasites during host cell invasion. PfRON2 possesses a region displaying homology with the rhoptry body protein PfRhopH1/Clag, a component of the RhopH complex. However, here we present co-immunoprecipitation studies which suggest that PfRON2 is not a component of the RhopH complex and has an independent role. Nucleotide polymorphism analysis suggested that PfRON2 was under diversifying selective pressure. This evidence suggests that RON2 appears to have a fundamental role in host cell invasion by apicomplexan parasites, and is a potential target for malaria intervention strategies.

Metabolomics is an 'omics' approach that aims to analyze all metabolites in a biological sample comprehensively. The detailed metabolite profiling of thousands of plant samples has great potential for directly elucidating plant metabolic processes. However, both a comprehensive analysis and a high throughput are difficult to achieve at the same time due to the wide diversity of metabolites in plants. Here, we have established a novel and practical metabolomics methodology for quantifying hundreds of targeted metabolites in a high-throughput manner. Multiple reaction monitoring (MRM) using tandem quadrupole mass spectrometry (TQMS), which monitors both the specific precursor ions and product ions of each metabolite, is a standard technique in targeted metabolomics, as it enables high sensitivity, reproducibility and a broad dynamic range. In this study, we optimized the MRM conditions for specific compounds by performing automated flow injection analyses with TQMS. Based on a total of 61,920 spectra for 860 authentic compounds, the MRM conditions of 497 compounds were successfully optimized. These were applied to high-throughput automated analysis of biological samples using TQMS coupled with ultra performance liquid chromatography (UPLC). By this analysis, approximately 100 metabolites were quantified in each of 14 plant accessions from Brassicaceae, Gramineae and Fabaceae. A hierarchical cluster analysis based on the metabolite accumulation patterns clearly showed differences among the plant families, and family-specific metabolites could be predicted using a batch-learning self-organizing map analysis. Thus, the automated widely targeted metabolomics approach established here should pave the way for large-scale metabolite profiling and comparative metabolomics.

In situ transfer of a self-transmissible, antibiotic-multiresistant plasmid RP4 from a laboratory Escherichia coli strain C600 to indigenous activated sludge bacteria was investigated using filter mating. The transfer frequency of RP4 from the donor E. coli to the bacteria that was sampled from two wastewater treatment plants was 5.1x10(-2) to 7.5x10(-1) and 4.6x10(-3) to 1.3x10(-2)/potential recipient. The isolated transconjugants showed resistance to Ap, Km, and Tc and the presence of a plasmid with a similar size to RP4. The traG gene on RP4 was also detected from all transconjugants. Reverse-transfer experiments from the transconjugants to E. coli HB101 indicated that RP4 maintained self-transmissibility in the transconjugants. The transconjugant strains were dominant bacteria in activated sludge including Pseudomonas fluorescens, P. putida, and Ochrobactrum anthropi and minor populations of enteric bacterial strains including Citrobacter freundii, E. coli, Enterobacter cloacae, E. asburiae, and Klebsiella pneumoniae ssp. pneumoniae. The transconjugant strains K. pneumoniae ssp. pneumonia, E. cloacae, and E. asburiae had several naturally occurring plasmids. These results suggest that in situ transfer of plasmids and the exchange of antibiotic-resistant genes can occur between released and indigenous bacteria in activated sludge.

Objective: TUR-P for the treatment of BPH is not performed in cases with severe heart disease due to the risk of bleeding and hyponatremia etc. Besides, in cases with severe central nerve disease and dementia, TUR-P is not performed because patients cannot take postoperative rest. We report the efficacy and the safety of Photoselective Vaporization of the Prostate (PVP) using a high-power 80 W KTP (potassiumu-titayl-phosphate) in patients for whom TUR-P cannot be performed., Patients and Methods: 312 patients underwent PVP in our hospital. Of these 312 patients, 8 patients with severe heart disease (American Society of Anesthesiology score of 3 or greater) and 4 patients with severe dementia (Performance status of 3 or greater) were evaluated. We assessed peak flow rate and post-void residual urine volume preoperatively and at 3 months postoperatively, safety of the procedure, and complication rates. The mean age was 81 years old (range 67 to 94) and the mean prostate volume was 63.8 ml (range 19 to 120). Urethral catheter was used in 8 patients because of chronic urinary retention. PVP was performed with GreenLightPV and a saline solution was used for irrigation. Upon completion of the procedure an 18Fr Foley catheter was inserted without continuous bladder irrigation and removed next morning, as a rule., Result: In all 12 patients, PVP was performed successfully without any intraoperative complications. Mean operating time was 101 minutes (range 28 to 184), mean total laser energy was 336,853 J (range 74,396 to 550,000), and mean irrigation volume was 21 L (range 8 to 36). The hemoglobin value changed from mean 13.1 mg/dl preoperatively to 12.2 mg/dl postoperatively. Blood transfusions were not required and hyponatremia was not observed. In 1 case, the urethral catheter was removed 3 hours following surgery. In the other cases, the urethral catheter was removed next morning. All patients could urinate after catheter removal and were discharged on the day following operation. Mean peak flow rate increased to 18 ml/s and mean post-void residual urine decreased to 46.9 ml at 3 months. No postoperative complications (urinary retention, hematuria, urinary tract infection, etc.) were observed., Conclusion: PVP is safe and effective in high-risk patients who are not candidates for TUR-P.

To investigate the physiologic role of cytosolic 2-Cys peroxiredoxin of Plasmodium berghei (PbTPx-1), we infected the vector mosquito Anopheles stephensi with a parasite carrying a targeted knockout of pbtpx-1 (Prx-KO). The number of Prx-KO midgut oocysts at 14-15 days post-feeding (pf) was comparable to that of the parent strain (WT); however, the numbers of sporozoites that formed in midgut oocysts and accumulated in the salivary gland of Prx-KO-infected mosquitoes by 21 days pf were decreased to 10-20% and 3-10%, respectively, of those values in WT-infected mosquitoes. A higher frequency of DNA strand breaks was detected in Prx-KO oocysts than in WT oocysts. Sporozoites carrying the targeted disruption had reduced infectivity in mice; however, the knockout did not affect the ability of the sporozoite to reach the liver parenchyma and initiate exo-erythrocytic form (EEF) development. TPx-1 may be involved in development during exponentially multiplying stages, such as sporozoites and EEF.

One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 mug/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.

We studied the impact of combined transurethral resection of the prostate (TURP) and high intensity focused ultrasound (HIFU) for localized prostate cancer (CaP) to decrease side effects such as prolonged urinary voiding disturbance observed after HIFU treatment. Included in this study were 18 patients with clinically localized CaP indicated for HIFU just followed by TURP (TUR combination group). Complete response was defined in accordance with ASTRO consensus statement and negative sample in biopsies performed 6 months after the HIFU treatment. Prostate specific antigen (PSA) nadir, International Prostate Symptom Score (IPSS) and morbidity during follow-up of TUR combination group were compared with those of a control of 18 patients who took HIFU treatment alone (HIFU monotherapy group). No statistical significances on the values of preoperative parameters (PSA, prostate volume, Gleason score, and IPSS) between these two groups. The median follow-up duration was 10 (5-15) months in both groups. A statistically significant impact was observed between TUR combination group and HIFU monotherapy group on median catheter time (5 versus 13 days, P<0.0001), PSA nadir (0.096 ng/ml versus 0.430 ng/ml in median, P<0.05) and the evolution of the post-treatment IPSS (8 versus 13.5 in median, P<0.0003) at 3 months after treatment. Urethral stricture necessary for urethral dilation was noted in 1 patient (5.6%) in the TUR combination group while in 2 (11.1%) in the HIFU monotherapy group. CR was obtained in 88.9% in the TUR combination group and 83.3% in the HIFU monotherapy group. Our study suggests that the combination of TURP with HIFU treatment improves posttreatment urinary status without additional morbidity.

A complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Understanding the level of polymorphism and the evolutionary processes is important for advancements in both vaccine design and knowledge of the evolution of cell invasion in this parasite. In the present study, we sequenced the entire open reading frames of seven genes encoding the proteins of the PfRhopH complex (rhoph2, rhoph3, and five rhoph1/clag gene paralogs). We found that four rhoph1/clag genes (clag2, 3.1, 3.2, and 8) were highly polymorphic. Amino acid substitutions and indels are predominantly clustered around amino acid positions 1000-1200 of these four rhoph1/clag genes. An excess of nonsynonymous substitutions over synonymous substitutions was detected for clag8 and 9, indicating positive selection. The McDonald-Kreitman test with a Plasmodium reichenowi orthologous sequence also supports positive selection on clag8. Based on the ratio of interspecific genetic distance to intraspecific distance, the time to the most recent common ancestor of the clag2 and 8 polymorphisms was estimated to be 1.89 and 0.87 million years ago, respectively, assuming divergence of P. falciparum and P. reichenowi 6 million years ago. In addition to a copy number polymorphism, gene conversion events were detected for the rhoph1/clag genes on chromosome 3, which likely play a role in increasing the diversity of each locus. Our results indicate that a high diversity of the PfRhopH1/Clag multigene family is maintained by diversifying selection forces over a considerably long period.

A 58-year-old male presented to a clinic with general weakness. Right adrenal tumor was found by computed tomography and he was referred to our hospital. Imaging studies revealed right adrenal tumor (8 cm) with marked swelling of surrounding lymph nodes and synchronous left renal tumor (2 cm) that was weakly enhanced by contrast media. Needle biopsy of the left kidney proved to be clear cell type renal cell carcinoma (RCC) and the preoperative diagnosis was left RCC and right primary adrenal cancer with lymph node metastasis. We performed right adrenalectomy, lymph node dissection and left radical nephrectomy. Pathological findings of right adrenal tumor and lymph nodes were both metastatic adenocarcinoma, which was not consistent with RCC or adrenal-derived carcinoma. Then, we extensively reviewed preoperative radiological examinations and found a small lesion in the left upper lung. This lesion was attached to the mediastinal shadow and there was no obvious lymph node swelling around this lesion. According to pathological findings and an elevation of carcinoembryogenic antigen, the adrenal lesion was diagnosed as adrenal metastasis of lung adenocarcinoma.

Anthocyanins and betacyanins, two types of red pigment, have never been found to occur together in plants. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales. The accumulation of flavonols in the Caryophyllales suggests that the step(s) of anthocyanin biosynthesis from dihydroflavonols to anthocyanins could be blocked in the Caryophyllales. Dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) cDNAs were isolated from plants of the Caryophyllales. An enzyme activity assay showed that the Caryophyllales possess functional DFR and ANS. The expression profile revealed that DFR and ANS are not expressed in most tissues and organs except the seeds in Spinacia oleracea. Here, the promoter regions of DFR and ANS were isolated from S. oleracea. Analysis of DFR and ANS promoter sequences revealed several putative transcriptional factor-binding motifs. A yeast one-hybrid assay showed that Petunia hybrida AN2 (PhAN2) and JAF13 (PhJAF13), which were the regulators of anthocyanin synthesis in P. hybrida, could bind to the S. oleracea DFR and ANS promoters. However, the transient assay in Phytolacca americana cell cultures and leaves of S. oleracea showed that the promoters were not activated by ectopic expression of PhAN2 and PhJAF13, while the DFR and ANS promoters of Arabidopsis thaliana, an anthocyanin-producing species, were activated. One possible explanation for the lack of anthocyanins in the Caryophyllales is the difference in the promoter regions of DFR and ANS compared with those of anthocyanin-producing species.

The Plasmodium circumsporozoite protein/thrombospondin-related anonymous protein-related protein (CTRP) is expressed at the mosquito midgut ookinete stage and is considered to be a transmission-blocking vaccine candidate. CTRP is composed of multiple von Willebrand factor A (vWA) and thrombospondin type 1 domains in the extracellular portion of the molecule, and a short acidic cytoplasmic domain that interacts with the actomyosin machinery. As a means to predict functionally relevant domains within CTRP we determined the nucleotide sequences of CTRP from the Plasmodium vivax Sall and the Plasmodium yoelii 17XL strains and characterized the conservation of domain architectures and motifs across Plasmodium genera. Sequence alignments indicate that the CTRP 1st to 4th vWA domains exhibit greater conservation, and thereby are perhaps functionally more important than the 5th and 6th domains. This point should be considered for the development of a transmission-blocking vaccine that includes CTRP recombinant subunit. To complement previous cellular studies on CTRP, we further determined the expression and cellular localization of CTRP protein in P. vivax and P. yoelii.

Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.

The Plasmodium falciparum high molecular mass rhoptry protein ('PfRhopH') complex is important for parasite growth and comprises three distinct gene products: RhopH1, RhopH2 and RhopH3. We have previously shown that P. falciparum RhopH1 is encoded by either PFC0110w (clag3.2) or PFC0120w (clag3.1), members of the previously-named clag (cytoadherence-linked asexual gene) multigene family. In this report, we have further characterized rhoph1/clag members in terms of gene structure, transcription and protein expression. The cDNA sequences for all five rhoph1/clag members were determined, confirming previous in silico predictions of intron-exon boundaries. All member genes were transcribed in HB3 and 3D7 parasite lines, but clag3.2 was not transcribed in Dd2 parasites. The peak abundance of transcripts for all genes was observed during the late schizont stage. Antisera specific to Clag2 and Clag3.1 localized these proteins to the apical end of merozoites in segmented schizonts, and both proteins are found to be components of the PfRhopH complex. PfRhopH complex that was immunoprecipitated with anti-Clag9 antibody contained neither Clag2 nor Clag3.1, thereby suggesting that PfRhopH complexes contain only individual rhoph1/clag gene products. Since the PfRhopH complex binds the erythrocyte surface, and RhopH2 and RhopH3 are encoded by single copy genes, the RhopH1/Clag proteins may serve to confer some degree of specificity to the roles of the individual complexes.

The result of combining the ultrasound Coded Excitation method and an ultrasound contrast agent (UCA), the Coded Harmonic Angio (CHA) technique provides arterial images with exceptional spatial, temporal, and contrast resolution that are comparable to those produced by conventional digital subtraction angiography. The authors report on their experience with intraoperative ultrasound arteriography performed using the transdural CHA technique in three patients: one harboring a meningioma, another with a middle cerebral artery aneurysm, and a third with an arteriovenous malformation. The present study demonstrates how intraoperative cerebral ultrasound arteriography can be applied to assess the adequacy of neurovascular procedures without the presence of an experienced operator.

The tobacco Tto1 is one of the few active LTR-retrotransposons of plants, and its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. The expression of Tto1 RNA can also be activated by various stresses, including viral infection, wounding, and treatment with jasmonate, a signal molecule of plant defence responses. It is shown here that the Tto1 LTR promoter is responsible for a high level of expression in cultured tissues of transgenic tobacco plants. We demonstrate that a 13-bp repeated motif (TGGTAGGTGAGAT) in the LTR functions as a cis-regulatory element, which confers the responsiveness to tissue culture, wounding and methyl jasmonate. Fungal elicitors also activate the promoter containing multiple copies of the 13-bp motif. Expression mediated by the 13-bp motif is activated markedly by okadaic acid and moderately by K252a, so that both phosphorylation and dephosphorylation of proteins are possibly involved in the signalling pathways. Interestingly, the 13-bp motif contains a conserved motif, Box L (also called AC-I or H-box like sequence) which has been shown to be involved in the expression of phenylpropanoid synthetic genes. Moreover, extended homologies are found between promoters of Tto1 and an asparagus defence gene, AoPR1, suggesting a possibility that the ancient insertion of an ancestral Tto1-related retrotransposon has provided some of the promoter/regulatory sequences, including the 13-bp motif-related sequence, of the AoPR1 gene. Based on the structural and functional similarity between the two promoters, a possible evolutionary role of the regulatory sequences of LTR-retrotransposons is discussed.

The tobacco retrotransposon Tto1, one of a few active retrotransposons of plants, has been shown to be activated by tissue culture. Its transposition is regulated mainly at the transcriptional level. It is shown here that expression of Tto1 can be induced in leaves of tobacco by wounding stress. Exogenous supply of methyl jasmonate, which is known to be a potent inducer of certain wound-responsive genes in plants, also induces Tto1 RNA expression. Tto1 RNA was detected within 2 to 4 h after wounding/cutting treatment, and increased levels of Tto1 RNA were observed during subsequent incubation periods for 48 h. Expression of Tto1 RNA after cutting treatment was induced more significantly in young expanding leaves rather than in older mature leaves, suggesting that developmental or physiological factors may be required for the strong response to Tto1 transcription to wounding stimuli. Experiments with transgenic tobacco plants carrying the Tto1-LTR: beta-glucuronidase fusion gene (LTR:GUS) revealed that Tto1 actually contains cis-regulatory regions in response to wounding and methyl jasmonate. These findings are discussed in relation to the mechanism of transcriptional activation and the evolutionary role played by retrotransposons.

Restenosis following carotid endarterectomy is not a rare condition. Among 122 endarterectomies we experienced, five restenoses (4.1%) were encountered and treated by the second surgery. The present report clarifies the clinical profiles and pathological findings of restenosis following carotid endarterectomy. Mean age of restenosis group (59 years old) was not significantly different from the group without restenosis (62 years old). Average duration between the first endarterectomy and the second surgery was 17 months (8-30 months). Initial symptoms were transient ischemic attack in three sides, minor stroke in one side, and asymptomatic in one. Degree of stenosis was tight (> or = 90%) in two and moderate (70-89%) in three. It is interesting to note that no ulcer was noted in the first endarterectomy specimen. At surgery for restenosis, two cases had symptoms and another two cases were asymptomatic, though all had neck bruits. Four of five lesions were treated by short venous graft from common carotid artery to distal internal carotid artery and another lesion was treated by second endarterectomy and Dacron patch graft. Pathology was studied in four and all showed myointimal hyperplasia. Three of four restenosis tissues showed mutant form p53 by immunohistochemistry. The present study indicates that restenosis following carotid endarterectomy is not a rare status. Short venous bypass across the stenotic portion is the treatment of choice. Monoclonal growth of smooth muscle with mutant form p53 might be related to the restenosis.

Plasminogen activators (PAs) have been suggested to play a role in neuronal migration and glial cell proliferation in the developing CNS. Less is known, however, about the role of PAs in the mature nervous system. To elucidate the role of tissue type plasminogen activator (tPA) in the nervous system we used in situ hybridization to study the expression of tPA mRNA within the rat facial nucleus after facial nerve transection. We also studied the effect of MK-801 on tPA mRNA expression in order to investigate whether the previously reported N-methyl-D-aspartate (NMDA) receptor activation is involved in this model. tPA mRNA was expressed in the ipsilateral facial motoneurones from 6 h after injury. This expression continued for at least 2 weeks after facial nerve transection. Administration of MK-801 before axonal injury did not affect the expression of tPA mRNA in the facial nucleus. These data suggest that tPA might be involved in the regenerative process without NMDA receptor activation in mature facial neurones.

The complete nucleotide sequence of the tobacco retrotransposon Tto1, one of the few active retrotransposons of plants, was determined. The sequence analysis suggests that Tto1 carries all functions required for autonomous transposition through reverse transcription. Gene organization and the nature of the transcription product suggest that Tto1 uses a gene expression mechanism different from those employed by retroviruses and most retrotransposons to regulate Gag and Pol stoichiometry. Tto1 was introduced into rice to study its autonomous transposition in heterologous hosts. Transcription and transposition of Tto1 were observed in rice cells. To probe the autonomous transposition through reverse transcription, a modified Tto1 retrotransposon in which part of a reverse transcriptase gene was replaced with an intron-containing hygromycin resistance gene was constructed and introduced into rice cells. Loss of the intron was observed only when intact Tto1 was cotransfected. These results indicate that Tto1 can transpose autonomously through reverse transcription and that the host factors required for transposition are conserved among monocots (class Magnoliopsida; rice) and dicots (class Liliopsida; tobacco), which diverged approximately 200 million years ago. These findings are discussed in relation to the regulation and evolution of retrotransposons and the possible use of Tto1 as a molecular genetic tool.

Extrachromosomal DNA forms of Drosophila retrotransposons (RTn) and retroviruses have been extensively analyzed. However, no such analysis with plant RTn has been reported. Here, we report the analysis of extrachromosomal forms of the tobacco RTn Tto1. Tto1 is one of a few active RTn of plants and has been shown to be activated in tissue culture. Extrachromosomal circular DNA forms of Tto1, with one or two long terminal repeats (LTR), were found in cultured cells. Sequence analysis of the sites of circularization through joining two LTR showed that the junction between the LTR contains small deletions and/or insertions. The insertions are heterogeneous and do not show any homology to the Tto1 sequence. Similar insertions have been detected in the extrachromosomal circular forms of the copia element of Drosophila and suggested to be the result of excision of genomic copia. The structural features of the junctions found in Tto1 suggest that the insertions are produced by a mechanism other than excision. The potential mechanism of production of the extrachromosomal circular forms of Tto1 is discussed.

The platelet derived growth factor (PDGF) plays an important role for development of atherosclerosis. We therefore immunostained carotid atheroma specimens for PDGF. We also detected dividing cell species of the atheroma with in vitro labeling of bromodeoxyuridine (BUdR). Thirty specimens of carotid atheroma were obtained by endarterectomy and they were incubated for 3 hours with Dulbecco's modified Eagle medium/20% fetal calf serum culture medium containing BUdR/fluorodeoxyuridine (FUdR). They were ethanol-fixed, thin-sliced, and immunostained for BUdR, PDGF, smooth muscle actin and macrophage. The PDGF immunoreactivity was mainly detected in the macrophages of the subendothelial area, where BUdR-positive cells were present. Percentage of BUdR-positive cells in the atheroma specimens ranged from 3% to 15%. The BUdR-labeled small cells were mainly located in the subendothelial area, and they were identified as non-foamy macrophages by double immunostaining with anti-macrophage antibody. The results indicate that nonfoamy macrophages have potentials for cell division and they might play an important role for the development and growth of atheroma by secreting PDGF.

We examined the hypoxic tolerance phenomenon in vitro. Brief exposure to hypoxia induced the production of basic fibroblast growth factor (bFGF) mRNA and protein in rat cortical neurons and protected them from hypoxic injury. Cortical neurons were cultured from 18th-day rat embryos in a serum-free medium and subjected to brief (4 h) and/or prolonged (24 h) hypoxia. Neuronal damage was assessed by quantifying lactate dehydrogenase (LDH) activity in the medium. After brief hypoxia, LDH release was identical to that of the controls, whereas prolonged hypoxia caused a significant increase in LDH release, indicating neuronal death. However, if brief hypoxia was applied 2 days prior to the prolonged hypoxia, no increase in LDH release was observed. The bFGF mRNA expression was assessed with Northern blot and protein immunoreactivity with Western blot analysis. The brief period of hypoxia caused a 2.5-fold increase in bFGF mRNA and considerable bFGF protein expression 1 day later, but prolonged hypoxia caused increase in the expression of bFGF mRNA at 2 days and no protein expression until 3 days after the start of the hypoxia. When cells were subjected to prolonged hypoxia 2 days after brief hypoxia, however, no increase in bFGF mRNA was observed, while bFGF protein was expressed continuously. We also observed that exogenously applied bFGF reduced neuronal injury produced by prolonged hypoxia. The results obtained with this model suggest that brief hypoxia induces bFGF protein and thus tolerance to subsequent lethal hypoxia. Basic FGF might play a role as a tolerance-associated factor in this process. Thus, an in vitro model is useful for assessing the response of cortical neurons to hypoxic stress and for researching new factors related to ischemic tolerance.