An in vivo screening system to identify tumorigenic genes.

Screening for oncogenes has mostly been performed by in vitro transformation assays. However, some oncogenes might not exhibit their transforming activities in vitro unless putative essential factors from in vivo microenvironments are adequately supplied. Here, we have developed an in vivo screening system that evaluates the tumorigenicity of target genes. This system uses a retroviral high-efficiency gene transfer technique, a large collection of human cDNA clones corresponding to ~70% of human genes and a luciferase-expressing immortalized mouse mammary epithelial cell line (NMuMG-luc). From 845 genes that were highly expressed in human breast cancer cell lines, we focused on 205 genes encoding membrane proteins and/or kinases as that had the greater possibility of being oncogenes or drug targets. The 205 genes were divided into five subgroups, each containing 34-43 genes, and then introduced them into NMuMG-luc cells. These cells were subcutaneously injected into nude mice and monitored for tumor development by in vivo imaging. Tumors were observed in three subgroups. Using DNA microarray analyses and individual tumorigenic assays, we found that three genes, ADORA2B, PRKACB and LPAR3, were tumorigenic. ADORA2B and LPAR3 encode G-protein-coupled receptors and PRKACB encodes a protein kinase A catalytic subunit. Cells overexpressing ADORA2B, LPAR3 or PRKACB did not show transforming phenotypes in vitro, suggesting that transformation by these genes requires in vivo microenvironments. In addition, several clinical data sets, including one for breast cancer, showed that the expression of these genes correlated with lower overall survival rate.

Effects of supplemental oxygen on urinary 8-hydroxy-2'-deoxyguanosine levels in extremely low birth weight infants.

As the effects of supplementary oxygen on urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) are poorly understood, urinary 8-OHdG levels (ng/mg creatinine) were determined longitudinally on the postnatal day (PND) 1, 3, and 30 in 16 neonates with birth weight < 1000 g. No supplementary oxygen was required in 9 neonates during the first 24 h of life. Urinary 8-OHdG level on PND 1 was inversely correlated with birth weight in these 9 neonates (P = 0.0323) and was higher in four with birth weight < 750 g than five with birth weight > 750 g (41.0 ± 6.9 vs. 5.6 ± 2.7, respectively, P = 0.0200). Median urinary 8-OHdG on PND 1 of these 9 neonates was significantly lower than that of 7 neonates with oxygen (9.3 vs. 60.2, respectively), although there were no significant differences in clinical background, such as birth weight, between the two groups. Five of the 9 did not require supplemental oxygen at all during the first 30 days of life. Median urinary 8-OHdG levels were consistently significantly lower in the 5 neonates than in 11 neonates with oxygen transiently or persistently (9.3 vs. 54.6, 19.1 vs. 61.4, and 28.3 vs. 145 on PND 1, 3, and 30, respectively), although there were no differences in clinical background, such as birth weight, between the two groups. Urinary 8-OHdG on PND 30 was significantly positively correlated with supplemental oxygen dose on PND 30 (P < 0.0001), but not with birth weight in the 16 neonates. These results suggest that higher supplemental oxygen tension caused higher urinary 8-OHdG in this population.

DOSY study on dynamic catenation: self-assembly of a [3]catenane as a meta-stable compound from twelve simple components.

Synthesis of [2]catenane 6 has been successfully achieved by the combination of Pd complex 1 and pyridines 2 and 3 at a molar ratio of 2:1:1 in D20. A mixture of square molecule 4 (prepared from 1 and 2) and macrocycle 5 (obtained from 1 and 3), in which the final ratio of 1, 2, and 3 was kept 2:1:1 reorganizes in D2O/CD3OD (1:1) to form 6 within one day. However, the same mixture in D2O shows the formation of novel [3]catenane 7 along with the [2]catenane. In order to make 7, the theoretical ratio of components 1, 2, and 3 should be 3:1:2. Thus, deliberately maintaining such ratio of the above-mentioned molecules, a higher proportion of the [3]catenane is observed in D2O as found from 1H NMR spectra of the system. Reorganization of the twelve components to form [3]catenane is supported by studies with the DOSY method. This method is a first attempt to separate, from a mixture, either catenanes or any other supramolecular self-assembly structures. CSI-MS studies further support the assigned catenane super structures 6 and 7. All the results indicate that the [2]catenane is thermodynamically the most stable structure, while the [3]catenane is a meta-stable self-assembly.

Change in the flavor of black tea drink during heat processing.

Heat processing during canning is responsible for the change in flavor of black tea infusion. The quantitative change in the volatile components of the black tea infusion during heat processing is not sufficient for explaining the sensory evaluation. In this study, application of aroma extract dilution analysis using the volatile fraction before and after black tea (Darjeeling) samples were heat processed resulted in the detection of 10 odor-active peaks for which flavor dilution (FD) factors changed. Seven potent odorants were identified from these peaks by gas chromatography-mass spectrometry. Among these components, 3-methylbutanal (stimulus), methional (potato-like), beta-damascenone (sweet), dimethyl trisulfide (putrid), and 2-methoxy-4-vinylphenol (clove-like) showed the highest FD factors after heat processing of the black tea sample. Therefore, these odorants were the most important components involved in changing the black tea odor during heat processing. In addition, the precursor of beta-damascenone in black tea infusion was investigated, and 3-hydroxy-7,8-didehydro-beta-ionol was determined to be one of the beta-damascenone-generating compounds for the first time.

Beta,gamma-methylene ATP-induced cAMP formation in C6Bu-1 cells: involvement of local metabolism and subsequent stimulation of adenosine A2B receptor.

The mechanism underlying beta,gamma-methylene ATP (beta,gamma-MeATP)-induced cAMP elevation was investigated in rat glioma C6Bu-1 cells. Beta,gamma-MeATP increased forskolin-stimulated cAMP formation in a manner sensitive to both the P1 antagonist xanthine amine congener (XAC) and the P2 antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Adenosine deaminase (ADA; 1 U/mL), which abolished the adenosine-induced response, did not eliminate the beta,gamma-MeATP-induced response. However, combination of ADA with alpha,beta-methylene ADP (alpha,beta-MeADP), an ecto-5'-nucleotidase inhibitor, blocked the beta,gamma-MeATP-induced response. AMP, the substrate for ecto-5'-nucleotidase, also induced cAMP formation in a manner sensitive to XAC and alpha,beta-MeADP inhibition. However, the AMP-induced response was not blocked by PPADS. HPLC analyses revealed that adenosine was generated from beta,gamma-MeATP and AMP. In addition, alpha,beta-MeADP inhibited the conversion of beta,gamma-MeATP and AMP to adenosine, whereas PPADS blocked adenosine formation from beta,gamma-MeATP but not from AMP. [3H]Adenosine generated from [3H]AMP was preserved on the cell surface environment even in the presence of ADA. The mRNAs for ecto-phosphodiesterase/pyrophosphatase 1 (EC 3.1.4.1), ecto-5'-nucleotidase (EC 3.1.3.5) and adenosine A2B receptor were detected by RT-PCR. These results suggest that C6Bu-1 cells possess ecto-enzymes converting beta,gamma-MeATP to adenosine, and the locally accumulated adenosine in this mechanism efficiently stimulates A2B receptors in a manner resistant to exogenous ADA.

Experimental study on lipid and bilirubin metabolism after biliary drainage for obstructive jaundice.

BACKGROUND: The present experimental study was carried out to determine variations in bilirubin and lipid metabolism in obstructive jaundice after external biliary drainage alone and in combination with intraintestinal administration of the drained bile. MATERIALS AND METHODS: Variations in lipid and bilirubin metabolism were studied in adult mongrel dogs with obstructive jaundice treated by external biliary drainage (EBD) and EBD plus intraintestinal administration of autologous bile (IBD). RESULTS: There was no difference between these two groups in regard to the volume of excreted bile after drainage. The biliary concentration and total daily excretion of bilirubin were higher in the IBD group (P < 0.0001), but there was no intergroup difference in the rate of decrease of serum bilirubin. In regard to lipid metabolism, the levels of total cholesterol, triglyceride, and low-density lipoprotein were decreased after biliary drainage; improvement in lipid metabolism was more rapid in the EBD group. Although the triglyceride level was lower after drainage, the activity of HMG-CoA reductase, the level of high-density lipoprotein 3-C, and the carrier protein apolipoprotein A-I were increased after drainage, with more rapid improvement in the EBD group. CONCLUSIONS: To improve lipid metabolism in obstructive jaundice, external biliary drainage is superior in the early stages of treatment, while replacement by EBD plus intraintestinal administration of autologous bile may be advantageous in cases of prolonged use.

Identification of potent odorants in Japanese green tea (Sen-cha).

Application of aroma extract dilution analysis using the volatile fraction of a Japanese green tea (Sen-cha) sample resulted in the detection of 36 odor-active peaks with flavor dilution (FD) factors between 10 and 5000. Thirty-six potent odorants were identified from 36 odor-active peaks by gas chromatography/mass spectrometry (GC/MS) and/or the multidimensional GC/MS (MDGC/MS) system. Among these components, 4-methoxy-2-methyl-2-butanethiol (meaty), (Z)-1, 5-octadien-3-one (metallic), 4-mercapto-4-methyl-2-pentanone (meaty), (E,E)-2,4-decadienal (fatty), beta-damascone (honey-like), beta-damascenone (honey-like), (Z)-methyl jasmonate (floral), and indole (animal-like) showed the highest FD factors. Therefore, these odorants were the most important components of the Japanese green tea odor. In addition, 4-methoxy-2-methyl-2-butanethiol, 4-mercapto-4-methyl-2-pentanone, methional, 2-ethyl-3, 5-dimethylpyrazine, (Z)-4-decenal, beta-damascone, maltol, 5-octanolide, 2-methoxy-4-vinylphenol, and 2-aminoacetophenone were newly identified compounds in the green tea.

Clinicopathological features of Churg-Strauss syndrome-associated neuropathy.

We assessed the clinicopathological features of 28 patients with peripheral neuropathy associated with Churg-Strauss syndrome. Initial symptoms attributable to neuropathy were acute painful dysaesthesiae and oedema in the dysaesthetic portion of the distal limbs. Sensory and motor involvement mostly showed a pattern of mononeuritis multiplex in the initial phase, progressing into asymmetrical polyneuropathy, restricted to the limbs. Parallel loss of myelinated and unmyelinated fibres due to axonal degeneration was evident as decreased or absent amplitudes of sensory nerve action potentials and compound muscle action potentials, indicating acute massive axonal loss. Epineurial necrotizing vasculitis was seen in 54% of cases; infiltrates consisted mainly of CD8-positive suppressor/cytotoxic and CD4-positive helper T lymphocytes. Eosinophils were present in infiltrates, but in smaller numbers than lymphocytes. CD20-positive B lymphocytes were seen only occasionally. Deposits of IgG, C3d, IgE and major basic protein were scarce. The mean follow-up period was 4.2 years, with a range of 8 months to 10 years. Fatal outcome was seen only in a single patient, indicating a good survival rate. The patients who responded well to the initial corticosteroid therapy within 4 weeks regained self-controlled functional status in longterm follow-up (modified Rankin score was < or = 2), while those not responding well to the initial corticosteroid therapy led a dependent existence (P < 0.01). In addition the patients with poor functional outcomes had significantly more systemic organ damage caused by vasculitis (P < 0.05). Necrotizing vasculitis mediated by cytotoxic T cells, leading to ischaemic changes, appears to be a major cause of Churg-Strauss syndrome-associated neuropathy. The initial clinical course and the extent of systemic vasculitic lesions may influence the long-term functional prognosis.

Clinicopathological features of chronic inflammatory demyelinating polyradiculoneuropathy in childhood.

The clinical, electrophysiological, and pathological findings, and the therapeutic characteristics in ten children with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), whose onset age was less than 16 years, were evaluated. The clinical progression pattern of the initial phase divided the patients into two groups. One group (six patients) showed a subacute progression for up to 2 months after onset and a subsided progression over 3 months. Three patients in this group had a preceding febrile episode. The other group (four patients) showed a chronic insidious progression for more than 3 months. The former group of patients revealed a favourable response to corticosteroid therapy as compared with the latter group. However, other clinical and laboratory features at the peak impairment were not distinguishable between these two groups. Motor dominant neuropathy was common to all patients, and only three cases showed sensory disturbance on the distal limbs. No cases revealed cranial nerve involvement. Motor and sensory nerve conduction and sural nerve biopsy studies revealed the demyelinating nature of the neuropathy. These clinicopathological features suggest that the subacute progression form frequently associated with prodromal episode and rather favourable corticosteroid response is characteristic in childhood CIDP, while the chronic insidious progression form is indistinguishable from the common adult CIDP.

[Double filtration plasmapheresis (DFPP) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP)].

We studied the therapeutic characteristics of double filtration plasmapheresis (DFPP) in 14 patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). The patients were classified into 2 subgroups of the responders (7 patients) and the non-responders (7 patients) to DFPP. The responders to DFPP were designated as those showing the improvement 2 or more grades in measures the activity of daily living by the modified Rankin scale (MRS). All these patients underwent neurological assessment, CSF study, electrophysiological studies at the beginning and end of treatment. Sural nerve biopsy study was performed in 10 cases. Neurological function was assessed serially using a quantitative neurological disability score (NDS). At the beginning of treatment, there were no significant differences in various measurements between the responders and the non-responders except for the frequency of demyelination. In responders, significant improvement was found in mean changes in MRS, NDS, motor nerve conduction velocity, compound muscle action potential, distal motor latency, while in non-responders, all measures remained unchanged or worsened. Muscle wasting was seen in 3/7 responders and 4/7 non-responders, and denervation potentials in needle EMG were seen in 1/7 responders and 3/7 non-responders. Four patients of the responders were classified as chronic relapsing course, and 6 patients of the non-responders as chronic progressive course. We conclude that DFPP was useful for the subgroups of CIDP patients, but the underlying immuno-pathological background that determine the efficacy of plasmapheresis should be elucidated.

[Serial MRI findings in patients with CNS cryptococcosis].

We reported the serial magnetic resonance imaging (MRI) findings of two patients with central nervous system (CNS) cryptococcal infection without AIDS. The diagnosis of CNS cryptococcosis was made by visualizing the fungi in the CSF with the India ink test, detecting cryptococcal antigens, and culturing the fungus. Both patients had dilated perivascular Virchow-Robin (V-R) spaces, which were defined as small rounded lesions greater less than 3mm diameter that were hyperintense on T2-weighted images. They were present in the basal ganglia, brainstem and cerebral white matter. Case 1 had bilateral parietal arachnoid cyst which was thought to represent a focal collection of organisms and mucoid material within subarachnoid space. Abnormal optochiasmatic arachnoid enhancement detected in case 2, who had complete loss of vision. With disease progression perivascular V-R increased in size, resulting in the developing cryptococomas which were defined as rounded lesions greater than 3mm diameter, and were hyperintense on T2-weighted images in the basal ganglia, cerebellum and cerebral white matter. In follow-up MRI of those patients, radiological progression was seen despite appropriate treatment and falling CSF cryptococcal antigens. In conclusion, this spectrum of MRI appearances in CNS cryptococcosis reflects the pathological mechanism of invasion by the fungus, and may be relatively specific for cryptococcosis.

Impairment of calcitonin gene-related peptide-induced potentiation of cholinergic sweat secretion in patients with multiple system atrophy.

Loss of sweating is a characteristic feature of multiple system atrophy (MSA) with autonomic failure, and widespread anhidrosis may lead to hyperthermia and collapse in a hot environment. Calcitonin gene-related peptide (CGRP) is present in the periglandular nerves around sweat glands and is a strong stimulant of methacholine (MCH)-mediated cholinergic sweating. The present study evaluated CGRP-related regulation of cholinergic sweating in patients with MSA. CGRP-induced potentiation of MCH-mediated cholinergic sweating was significantly reduced in MSA patients as compared with normal age-matched controls. These results suggest that regulation of sweating is extensively affected in MSA as a consequence of peptidergic dysfunction.

Possible mechanism of anhidrosis in a symptomatic female carrier of Fabry's disease: an assessment by skin sympathetic nerve activity and sympathetic skin response.

Fabry's disease, X-linked alpha-galactosidase deficiency, features a variety of autonomic abnormalities including anhidrosis. In this study, we measured the skin sympathetic nerve activity (SSNA), skin potential and sweat rate in a symptomatic female carrier to investigate the underlying pathophysiology of anhidrosis. The basal activity and responsiveness of SSNA were both fairly well preserved, although slightly reduced compared with the control levels. However, sweating was completely absent, despite the normal skin potential change in response to SSNA bursts. These results suggest that anhidrosis in Fabry's disease is a result of sweat gland dysfunction as well as abnormal SSNA.

Variant of intraductal carcinoma (with scant mucin production) is of main pancreatic duct origin: a clinicopathological study of four patients.

OBJECTIVES: To investigate the histopathological and immunohistochemical features of intraductal "nodular" tumor located in the main pancreatic duct. METHODS: Four cases were studied, and the results were compared with those in 10 cases of common ductal adenocarcinomas of the pancreas. RESULTS: The intraductal nodular carcinoma were large, more than 3 cm in diameter (range, 3-7 cm), lacked macroscopic mucin production, and presented a papillotubular adenocarcinoma. These tumors showed minimal invasion into the periductal tissue, duodenum, and choledochus. Immunohistochemically, the tumor cells were negative for anti-CEA and faintly positive for anti-CA19-9, in all except one tumor, whereas the common ductal adenocarcinomas were clearly positive for both. The postoperative course was favorable, and all three living patients remained healthy for more than 4 yr. CONCLUSION: These tumors may be variants of intraductal tumors with a more favorable prognosis.

[Peptidergic sweating in multiple system atrophy and hereditary spinocerebellar ataxia with special reference to calcitonin gene-related peptide].

Calcitonin gene-related peptide (CGRP)-like immunoreactivity has been demonstrated to be present in nerve terminals around the sweat glands. We have previously demonstrated that CGRP modulates the cholinergic sweating activity. The present study was designed to evaluate the locally administrated CGRP on sweating of patients with multiple system atrophy (MSA) and hereditary spinocerebellar ataxia (HSCA) consisting of 10 males and 11 females. Among 9 HSCA, 3 was diagnosed as Machado-Joseph disease by clinical and DNA diagnostic assessment. CGRP and methacholine chloride (MCH) was dissolved in saline solution to specified concentration, and 0.1ml of which was injected intradermally at the center of a forearm test area. The sweat rate was recorded continuously by the capacitance hygrometry in a relatively cool environment in which the spontaneous sweating was absent. CGRP significantly increased the sweat rate when it was administrated with MCH on normal subjects. There was a clear dose-dependent relationship between the dose of CGRP and its enhancement. This enhancement was significantly reduced in patients with MSA as compared with controls. On the other hand, most of HSCA showed no remarkable impairment of CGRP enhancement. These results suggest that peptidergic sweating is extensively affected in patients with MSA but is not in patients with HSCA, which may be the consequence of peptidergic sweating dysfunction in MSA.

[Phenotypic heterogeneity in Japanese Charcot-Marie-Tooth disease type 1A patients with PMP-22 gene duplication].

We studied phenotypic heterogeneity in 18 Japanese patients with Charcot-Marie-Tooth disease type 1A (CMT1A) with PMP-22 gene duplication, together with heterogeneity of duplication size. In order to detect the duplication of PMP-22 gene region, the PMP-22 cDNA and a polymorphic marker VAW409R3 were used as probes for Southern blot analysis. As the clinical phenotypes, we assessed the degree of foot deformity, muscular weakness and atrophy, tendon reflexes, sensory impairment and electrophysiologic and sural nerve biopsy findings. Although the degree of muscular weakness and atrophy was slightly more severe in the advanced age, there was a patient with calf hypertrophy in the older age or a patient with marked muscular atrophy of the leg in the younger age. The incidence of foot deformities and sensory impairment was high and these phenotypes did not relate to aging. Diminished or absent tendon reflexes and slowing of motor conduction velocities were commonly seen, but the motor conduction velocity varied greatly among the patients. The clinical phenotypes were extensively variable among the CMT 1A patients with the same gene mutation of PMP-22 gene duplication, suggesting that there is a factor other than PMP-22 gene duplication, which influences phenotypic manifestation in CMT 1A.

[Sensory ataxic neuropathy associated with Sjögren's syndrome].

One tenth of patients with Sjögren's syndrome (SS) is associated with peripheral neuropathy. Trigeminal neuropathy, mononeuropathy multiplex, sensory neuropathy, distal sensory neuropathy, ataxic sensory-autonomic neuropathy have been described as the neuropathy associated with SS. However, some of them secondary occur associated with the vasculopathy due to rheumatoid arthritis, systemic lupus erythematodes etc combined with SS. We demonstrated the clinico-patho-physiological features and spinal MRI findings of patients with primary SS in whom the sensory ataxia and autonomic dysfunctions were the predominant symptoms. The underlying pathology was lymphocytic (T cells) infiltration to the dorsal root ganglia with sensory neuronal degeneration. These findings have identified as association between SS and sensory ataxic neuropathy.

[Abnormality of PMP-22 gene in Japanese patients with Charcot-Marie-Tooth disease--comparison between Southern blot and polymerase chain reaction analysis in the detection of PMP-22 gene duplication].

We investigated the duplication of the PMP-22 gene in 23 Japanese patients with Charcot-Marie-Tooth disease (CMT) by Southern blot and PCR analysis. To detect duplication of the PMP-22 gene region, PMP-22 cDNA and a polymorphic marker, VAW409R3, located in the region flanking the PMP-22 gene, were used as probes for Southern blot analysis. A marker, 6G1, also located in the PMP-22 flanking region, was amplified using the quantitative PCR method. The signal intensity of the 2.8-kb and 2.7-kb bands of MspI digests probed with VAW409R3 was different in patients with duplication. The ratio of these two bands, measured by densitometry, ranged from 1.75 to 2.13 in the patients with duplication and from 1.01 to 1.15 in those without duplication and in normal controls. The signal ratio of PMP-22 to the reference marker SF85 of BamHI digests ranged from 1.15 to 1.33 in the patients with duplication and from 0.96 to 1.04 in those without duplication, when compared with normal controls assigned a value of 1.0. The ranges of the intensity in these two groups were narrow, but did not overlap. The signal ratio of the PCR products of 6G1 to reference marker D1S80 on quantitative PCR analysis was also measured. The ratio ranged between 1.67 and 2.23 with duplication and between 1.29 and 1.74 without duplication according to the results of Southern blot analysis. However, some patients exhibited overlapping signal intensity in the duplication and non-duplication ranges. Thus, these three methods each has its own advantages and disadvantages in regard to detecting duplication.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma exchange in chronic inflammatory demyelinating polyradiculoneuropathy; different beneficial effects and their correlation to the clinical features.

We report 7 patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) who showed significant clinical improvement by plasma exchange (PE). Their clinical features were extensively varied but fulfilled the diagnostic criteria for CIDP. The patients could be divided into 2 subgroups based on the mode of effectiveness of PE; one group consisted of those who once achieved complete remission, but required highly frequent PEs for long-term maintenance of remission and the others were those who showed persistent and complete remission with several sessions of PE alone. These findings raise issues about the long-term therapeutic goals to be achieved by PE for CIDP patients, particularly on supportive therapy other than PE, as well as the optimization of frequency and duration of PE.