The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme.

Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.

A Systematic Approach to Preclinical Trials in Metastatic Breast Cancer.

The process of developing new agents for therapy against breast cancer is inefficient and relies on animal models to screen for efficacy for preclinical studies. However, there has been limited validation of these models, despite the increasing costs in the rapidly growing era of personalized medicine and targeted therapy. Recently, there have been multiple studies which have critically evaluated animal models for breast cancer drug discovery. We recently reviewed the transgenic, xenograft, and syngeneic murine breast cancer models, the ectopic, orthotopic and intravenous methods of cell implantation, tumor gene expression profiles, as well as the ethics of animal experimentation, and we provide important information for investigators in this challenging field. Because of the complexities of treating breast cancer and the increasing costs of developing new agents, the choice of the appropriate murine model must carefully consider each model available, including the tumor gene expression profile. Such a critical approach to the in vivo portion of drug development will further increase the efficiency of breast cancer drug research and development.

Roles of pathway-specific and inhibitory Smads in activin receptor signaling.

Activins and other members of the transforming growth factor-beta-like superfamily of growth factors transduce their signals by interacting with two types of receptor serine/threonine kinases. The Smad proteins, a new family of intracellular mediators are involved in the signaling pathways of these receptors, but the initial stages of their activation as well as their specific functions remain to be defined. We report here that the pathway-specific Smad2 and 3 can form a complex with the activin receptor in a ligand-dependent manner. This complex formation is rapid but also transient. Indeed, soon after their association with the activin receptor, Smad2 and Smad3 are released into the cytoplasm where they interact with the common partner Smad4. These Smad complexes then mediate activin-induced transcription. Finally, we show that the inhibitory Smad7 can prevent the association of the two pathway-specific Smads with the activin receptor complex, thereby blocking the activin signal.

The phosphorylated prodrug FTY720 is a histone deacetylase inhibitor that reactivates ERα expression and enhances hormonal therapy for breast cancer.

Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer.

Interruption of activin A autocrine regulation by antisense oligodeoxynucleotides accelerates liver tumor cell proliferation.

Administration of activin A, a member of the transforming growth factor-beta superfamily inhibits hepatocyte proliferation in vitro and reduces liver mass in vivo. However, a role of endogenous activin A in local growth modulation has not been established in any system. The aim of this study was to examine the production of activin A in the human hepatoma cell line HLF and to explore a possible autocrine role of activin as a cell growth inhibitor by blocking production of endogenous activin using antisense oligodeoxynucleotides. Administration of exogenous activin A suppressed HLF cell growth, and immunoreactive activin A was shown to be produced in the cells at confluency by Western blotting analysis. Cells were exposed to phosphorothioate-modified oligodeoxynucleotides, synthesized with antisense or randomly shuffled base sequences of activin betaA subunit messenger RNA, under serum-free conditions. Uptake of the oligodeoxynucleotides into the cells was confirmed by use of fluorescein isothiocyanate-labeled oligodeoxynucleotides. Administration of antisense oligodeoxynucleotides reduced activin A production as confirmed by both competitive PCR and Western blotting. Activin betaA antisense oligodeoxynucleotides significantly increased cell proliferation compared with controls. These findings are consistent with the existence of an autocrine role of activin A as an inhibitor of hepatocyte proliferation.

Production of fungal fructosyl amino acid oxidase useful for diabetic diagnosis in the peroxisome of Candida boidinii.

A high-level production of fructosyl amino acid oxidase (FAOD), whose production was toxic in Escherichia coli, was investigated through attempts to utilize the peroxisome of Candida boidinii as the place for protein accumulation. The alcohol oxidase-depleted strain (strain aod1Delta) produced FAOD at a four to five times higher level than the wild type strain in terms of protein amount and enzyme activity, although the transcriptional level was similar. As a result of this study, we could improve FAOD productivity approximately 47-fold from the original transformant, and FAOD accumulated within membrane-bound peroxisomes up to 18% of the total soluble proteins.

Minute squamous cell carcinoma arising in the wall of a congenital lung cyst.

A case of minute squamous cell carcinoma arising in a congenital lung cyst is reported. Two aspects of this case were notable. First, we were able to make the diagnosis of squamous cell carcinoma by bronchial lavage at an early stage. Second, to our knowledge, this is the first case of probable de novo origin of squamous cell carcinoma in a congenital lung cyst.

Thoracoscopic diagnosis and treatment of chylothorax after pneumonectomy.

Chylothorax after pneumonectomy was treated successfully by selective application of fibrin glue through a thoracoscope. Conservative therapy for 18 days failed to close the fistula. The site of leakage was identified during thoracoscopy, and fibrin glue was applied under direct vision. Leakage decreased immediately and eventually stopped completely. This technique is less invasive than standard surgical treatment and should be considered in all patients with postoperative chylothorax.

Carney's complex in association with right atrial myxoma.

The association between myxomas, spotty pigmentation, and endocrine overactivity is referred to as Carney's complex. This report describes the case of a 24-year-old woman with right atrial myxoma who presented with this association. The patient had a previously excised myxoid tumor of the breast and uneven facial pigmentation was observed, but no evidence of endocrinopathy was detected. The myxoma originated from the right atrial wall adjacent to the junction of the inferior vena cava and right atrium, and was successfully excised. A brief review of Carney's complex is provided after discussion of this rare case.

Subcellular localization of fructosyl amino acid oxidases in peroxisomes of Aspergillus terreus and Penicillium janthinellum.

Fructosyl amino acid oxidase (FAOD) is the enzyme catalyzing the oxidative deglycation of Amadori compounds, such as fructosyl amino acids, yielding the corresponding amino acids, glucosone, and H(2)O(2). In a previous report, we determined the primary structures of cDNAs coding for FAODs from two fungal strains Aspergillus terreus AP1 and Penicillium janthinellum and we found that both fungal FAODs included the putative peroxisome targeting signal 1 (PTS1) at the carboxyl terminal (Yoshida, N. et al., Eur. J. Biochem., 242, 499-505, 1996). In this study, we determined the intracellular localization of FAODs in these two fungi. Subcellular fractionation experiments and immuno-electronmicroscopic observations, together with the previous findings indicated that the FAODs were localized in peroxisomes of A. terreus AP1 and P. janthinellum. These FAODs were also found to belong to a new member of "peroxisomal sarcosine oxidase family protein" in eucaryotic cells.

Segmental pericholangial fibrosis: a peculiar benign fibrosing disease at the hepatic hilum.

The authors report on a 9-year-old child who underwent surgery to remove a tumor of the hepatic hilum with preoperative radiographic studies suggestive of malignancy, but whose surgical specimens showed a peculiar fibrosing disease. The lesion was localized to the bifurcation of the hepatic duct, where the bile duct wall and the surrounding tissue was markedly fibrotic. No malignant cells or epithelial destruction were seen. The patient's postoperative course was uneventful, and he is without any sign of recurrence 2 years after surgery. Because the histological features of this case do not correspond to any established disease, including primary sclerosing cholangitis, the authors believe it represents a new entity, segmental pericholangial fibrosis. Local resection resulted in a good outcome. A review of the literature disclosed a few similar cases with a benign clinical course.

A new variant of alpha-1-antitrypsin deficiency (Siiyama) associated with pulmonary emphysema.

A 38-year-old male with pulmonary emphysema due to severely reduced serum alpha-1-antitrypsin (AAT) level (14.5 mg/dl) was found to have an inherited new AAT deficient variant Siiyama. Chest roentgenogram and CT scanning revealed advanced emphysema, and severe obstructive ventilatory impairment was observed. During the 4-year follow-up period, the annual rate of decline of FEV 1.0 showed approximately 10-fold greater than the normal decline in FEV 1.0 (-380 ml/yr). Treatment with tamoxifen in order to raise the serum AAT level only resulted in an insufficient increase. Augmentation therapy of human AAT should be considered in the future.

Computed tomography (CT) findings of gastric rupture after blunt trauma.

A 49-year-old carpenter was hit by timber around his upper abdomen 1 hour after breakfast. Immediate computed tomography was taken, followed by emergency laparotomy showing gastric rupture accompanied with hemorrhage from the superior mesenteric vein. Hemostasis and distal partial gastrectomy followed by Billroth-I anastomosis reconstruction was performed. Here, we report the abdominal computed tomography findings from a patient with gastric rupture after blunt trauma. The present case, which is only the second such case reported in English literature, suggested that computed tomography is useful for assessing associated injuries in gastric rupture patients, for detecting intraperitoneal free air which can be missed by X-rays, and for locating the laceration of the rupture.

Beta(+)-thalassemia with hemochromatosis.

A 64-year-old man was admitted due to ascites. Laboratory data showed hemoglobin 6.7 g/dl, mean corpuscular volume 82 fl, and ferritin 2,360 ng/ml. Liver biopsy showed hemochromatosis. The diagnosis of beta-thalassemia was suggested by a decreased ratio of beta/alpha-globin synthesis in vitro (0.26). Cloning of the beta-globin gene showed A-to-G mutation in the first base of the ATA box. He was confirmed to be homozygous for this specific allele by beta-gene complex analysis and analysis of Southern blot hybridization of the alpha- and beta-globin genes. His two sons were confirmed to be heterozygous for this allele.

Malignant lymphoma involving the penis following malignant pleural mesothelioma.

A 74-year-old man who had been diagnosed with malignant mesothelioma developed malignant lymphoma of B-cell origin involving the penis. He had a history of occupational exposure to asbestos as a construction worker. The association of malignant mesothelioma with lymphoma is rare, and the possibility of asbestos exposure as a common etiology is discussed. The intense stimulation of B lymphocytes and decreased T lymphocyte activity in asbestos-exposed populations may result in development of B-cell malignancies. Though the relationship between asbestos exposure and malignant mesothelioma is firmly established, the relationship between asbestos exposure and lymphoma remains to be investigated.

[Light and electron microscopic observations of cysts in the brain from an autopsy case of cryptococcus meningitis].

We gave some considerations to the significance of cyst formation in a case of cryptococcus meningitis by examining the cysts themselves and comparing the organisms in the cysts with those disseminated throughout the subarachnoid space by light and electron microscopy. An 18-year-old girl had complained of headache, stiffneck and fever at the onset. These symptoms worsened into confusion without any definite diagnosis, then resulted in an arrest of spontaneous respiration which led to use of respirator for 12 days. The patient died 40 days after the onset. The brain weighed 1440 g and showed moderate swelling with opacity of the leptomeninges, which was very evident over the convexity and around the basal side of the pons. Subarachnoid fresh hemorrhage was also observed around the basal side of the brain stem. Microscopic examination of the subarachnoid space revealed widely disseminated Cryptococcus neoformans varied in size, whose cell wall showed a positive staining reaction to PAS. The organisms had characteristic spicules positively stained with cresyl violet radiating out of the cell body, and were associated with infiltration of lymphocytes, macrophages and polymorphonuclear leukocytes throughout the subarachnoid space. Some portions of arachnoid membrane, dura mater and vessel walls in the subarachnoid space especially the adventitia of the basilar artery were replaced by multiple cysts. The cysts were tightly filled with large numbers of small uniformly sized organisms which often showed budding. These cysts showed no histological evidence for inflammation. Further studies to demonstrate those differences were carried out with electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of lung cancer with mediastinal tumor].

A 72-year-old female underwent surgical resection of lung cancer and mediastinal tumor. Abnormal shadows on the chest roentgenogram were incidentally detected by the regular health screening. The lung cancer existed in the left upper lobe, and the mediastinal tumor existed in the posterior mediastinum along left side of the spines. Serum CEA level was 299 ng/ml by enzyme immunoassay method. We judged the lung cancer as being in Stage I (T2 N0 M0), and the mediastinal tumor as benign neurogenic tumor unrelated to the lung cancer through detailed examination. Surgical resection of both tumors was performed through left thoracotomy, and the surgical procedure was curative operation. Pathological examination revealed the lung cancer was moderately differentiated adenocarcinoma and the mediastinal tumor was Schwannoma. Eight months after surgery, serum CEA level is 1.1 ng/ml without evidence of recurrence. This experience suggested us that a mediastinal tumor accompanied by lung cancer is not necessarily metastatic tumor, and that curative operation may be possible in these cases.

Xylan deposition on secondary wall of Fagus crenata fiber.

Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited.

Autolysis during in vitro tracheary element differentiation: formation and location of the perforation.

Tracheary elements differentiated from isolated Zinnia: mesophyll cells were observed at various times of culture under a scanning electron microscope. Perforation occurred on the primary wall at one of the longitudinal ends in single tracheary elements. In double tracheary elements, which both of two cells derived from a single cell differentiated into, the pore opened on the primary walls both at the junction of the two tracheary elements and at a longitudinal end of one of the two tracheary elements. These results suggest not only that a single tracheary element has its own program to form a perforation at one end without being affected by neighboring cells, but also that isolated cells indeed hold some traces of polarity and cell-cell communication.