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We here taxonomically revise the suborder Massarineae(Pleosporales, Dothideomycetes, Ascomycota). Sequences of SSU and LSU nrDNA and the translation elongation factor 1-alpha gene (tef1) are newly obtained from 106 Massarineaetaxa that are phylogenetically analysed along with published sequences of 131 taxa in this suborder retrieved from GenBank. We recognise 12 families and five unknown lineages in the Massarineae. Among the nine families previously known, the monophyletic status of the Dictyosporiaceae, Didymosphaeriaceae, Latoruaceae, Macrodiplodiopsidaceae, Massarinaceae, Morosphaeriaceae, and Trematosphaeriaceaewas strongly supported with bootstrap support values above 96 %, while the clades of the Bambusicolaceaeand the Lentitheciaceaeare moderately supported. Two new families, Parabambusicolaceaeand Sulcatisporaceae, are proposed. The Parabambusicolaceaeis erected to accommodate Aquastromaand Parabambusicolagenera nova, as well as two unnamed Monodictysspecies. The Parabambusicolaceaeis characterised by depressed globose to hemispherical ascomata with or without surrounding stromatic tissue, and multi-septate, clavate to fusiform, hyaline ascospores. The Sulcatisporaceaeis established for Magnicamarosporiumand Sulcatisporagenera nova and Neobambusicola. The Sulcatisporaceaeis characterised by subglobose ascomata with a short ostiolar neck, trabeculate pseudoparaphyses, clavate asci, broadly fusiform ascospores, and ellipsoid to subglobose conidia with or without striate ornamentation. The genus Periconiaand its relatives are segregated from the Massarinaceaeand placed in a resurrected family, the Periconiaceae. We have summarised the morphological and ecological features, and clarified the accepted members of each family. Ten new genera, 22 new species, and seven new combinations are described and illustrated. The complete ITS sequences of nrDNA are also provided for all new taxa for use as barcode markers.

We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidaeis expanded to include the aquatic order Jahnulales.An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.

A new pleosporalean family Tetraplosphaeriaceaeis established to accommodate five new genera; 1) Tetraplosphaeriawith small ascomata and anamorphs belonging to Tetraploa s. str., 2) Triplosphaeriacharacterised by hemispherical ascomata with rim-like side walls and anamorphs similar to Tetraploabut with three conidial setose appendages, 3) Polyplosphaeriawith large ascomata surrounded by brown hyphae and anamorphs producing globose conidia with several setose appendages, 4) Pseudotetraploa, an anamorphic genus, having obpyriform conidia with pseudosepta and four to eight setose appendages, and 5) Quadricrura, an anamorphic genus, having globose conidia with one or two long setose appendages at the apex and four to five short setose appendages at the base. Fifteen new taxa in these genera mostly collected from bamboo are described and illustrated. They are linked by their Tetraploa s. l.anamorphs. To infer phylogenetic placement in the Pleosporales, analyses based on a combined dataset of small- and large-subunit nuclear ribosomal DNA (SSU+LSU nrDNA) was carried out. Tetraplosphaeriaceae, however, is basal to the main pleosporalean clade and therefore its relationship with other existing families was not completely resolved. To evaluate the validity of each taxon and to clarify the phylogenetic relationships within this family, further analyses using sequences from ITS-5.8S nrDNA (ITS), transcription elongation factor 1-α (TEF), and β-tubulin (BT), were also conducted. Monophyly of the family and that of each genus were strongly supported by analyses based on a combined dataset of the three regions (ITS+TEF+BT). Our results also suggest that Tetraplosphaeria(anamorph: Tetraploa s. str.) is an ancestral lineage within this family. Taxonomic placement of the bambusicolous fungi in Astrosphaeriella, Kalmusia, Katumotoa, Massarina, Ophiosphaerella, Phaeosphaeria, Roussoella, Roussoellopsis, and Versicolorisporium, are also discussed based on the SSU+LSU phylogeny.

AbstractBackground: Although hypertension is commonly found in patients with autosomal dominant polycystic kidney disease (ADPKD), there is no consensus about which antihypertensive agents are most appropriate. The effects of calcium channel blockers (CCB) and angiotensin II receptor blockers (ARB) on blood pressure and renoprotection were compared in hypertensive patients with ADPKD. Methods: We randomly assigned 49 participants to CCB amlodipine-based (2.5–10 mg/day) or ARB candesartan-based (2–8 mg/day) regimens. Twenty-five patients (13 males and 12 females) received amlodipine, and 24 patients (13 males and 11 females) received candesartan. This was followed up for 36 months. Results: Baseline characteristics were similar, and blood pressure was well controlled in both groups throughout the study period. Six out of 25 (24.0%) amlodipine and 1 out of 24 (4.2%) candesartan patients were terminated from the protocol due to a twofold increase in serum creatinine and/or decrease in creatinine clearance (Ccr) to half of the baseline. The renal event-free survival rate was significant (p < 0.05, Breslow-Gehan-Wilcoxon test). Serum creatinine was higher in the amlodipine group than in the candesartan group at 24 and 36 months (p < 0.05). The decrease in Ccr at 36 months was larger in the amlodipine group than in the candesartan group (ΔCcr: –20.9 ± 13.1 vs. –4.8 ± 13.8 ml/min, p < 0.01). Urinary protein excretion was significantly lower in the candesartan group than in the amlodipine group at 36 months. Urinary albumin excretion was significantly lower in the candesartan group than in the amlodipine group at 12, 24 and 36 months. Conclusions: The renoprotective effect of candesartan is considered more favorable than amlodipine in the treatment of ADPKD. This is independent of the antihypertensive effect per se.Copyright © 2005 S. Karger AG, Basel

AbstractObjective: Methylglyoxal (MGO) in a heat-sterilized conventional PD solution may damage peritoneal cells directly and/or indirectly by producing advanced glycation endproducts (AGEs). This study was conducted to (a) examine the acute effect of MGO on the peritoneum (including AGE formation) and (b) study the possible AGE suppressive effect of an anti-oxidant, sodium sulfite. Method: (1) Human serum albumin (HAS) was continuously incubated with MGO (50 mM) at 37°C for as long as 14 days and the fluorescence intensity (FI) was determined (em. 440, ex. 370). (2) Three types of test solutions – (i) saline; (ii) MGO (20 mM), and (iii) MGO with sodium sulfite (30 mM) – were administered intraperitoneally to 8-week-old rats once a day for 5 consecutive days. The parietal peritoneum was examined macroscopically on the 6th day for immunostaining of anti-AGE antibodies. Result: (1) An increase in FI of HSA was observed as a function of the incubation period in the MGO solution. (2) Prominent hypervascularity and intense immunostaining of anti-AGE Ab were noted in MGO-treated rats, whereas the macroscopic alterations were suppressed in the rats that had been treated with sodium sulfite. Conclusion: MGO-induced hypervascularity and AGE formation in the peritoneum, as well as macroscopic alterations were suppressed by sodium sulfite. This may indicate that there is a risk of MGO causing a peritoneal injury and that the therapeutic potential of an anti-oxidant for this type of injury may exist.Copyright © 2003 S. Karger AG, Basel

Cardiovascular disease is a major problem in end-stage renal disease (ESRD) patients, with calcification being one of the conspicuous features of arteriosclerotic vessels. In the present study, clinical analysis and in vitro cell culture were used to investigate factors promoting vascular calcification in ESRD patients. The aortic arch calcification score (AACS) was the method used to estimate vascular calcification by evaluation of the simple posterior-anterior view chest X-rays. Factors that relate significantly to vascular calcification and the AACS are the Ca×Pi, age, dialysis period, blood pressure, smoking and diabetes mellitus, but not total cholesterol or triglyceride. The Ca×Pi, which depends on the serum phosphate concentration, is the only specific factor with the possibility for correction in ESRD patients, and so control of serum phosphate concentration is an important factor for reducing vascular calcification. The effects of phosphate overload on calcium deposition in human vascular smooth muscle cells (hVSMCs) using a primary cell culture system were also investigated. hVSMCs were harvested from the radial artery in ESRD patients and it was found that they could secrete extracellular matrix with a high affinity for calcium in a high phosphate medium (Pi=5.4 mg/dl). Therefore, phosphate overload might stimulate the hVSMCs to accelerate the calcium deposition in ESRD patients. These results suggest that the control of phosphate excess is important for prevention of calcium deposition on arteriole walls in ESRD patients.

Cardiovascular disease is a major problem in end-stage renal disease (ESRD) patients, with calcification being one of the conspicuous features of arteriosclerotic vessels. In the present study, clinical analysis and in vitro cell culture were used to investigate factors promoting vascular calcification in ESRD patients. The aortic arch calcification score (AACS) was the method used to estimate vascular calcification by evaluation of the simple posterior-anterior view chest X-rays. Factors that relate significantly to vascular calcification and the AACS are the Ca×Pi, age, dialysis period, blood pressure, smoking and diabetes mellitus, but not total cholesterol or triglyceride. The Ca×Pi, which depends on the serum phosphate concentration, is the only specific factor with the possibility for correction in ESRD patients, and so control of serum phosphate concentration is an important factor for reducing vascular calcification. The effects of phosphate overload on calcium deposition in human vascular smooth muscle cells (hVSMCs) using a primary cell culture system were also investigated. hVSMCs were harvested from the radial artery in ESRD patients and it was found that they could secrete extracellular matrix with a high affinity for calcium in a high phosphate medium (Pi=5.4 mg/dl). Therefore, phosphate overload might stimulate the hVSMCs to accelerate the calcium deposition in ESRD patients. These results suggest that the control of phosphate excess is important for prevention of calcium deposition on arteriole walls in ESRD patients.

Abstract:  Hyaloscypha albohyalina var. monodictys, a new variety in the family Hyaloscyphaceae, Helotiales with Monodictys anamorph is described and illustrated.

Acellular haemoglobin (Hb) has intrinsic toxicity to the tissues since harmful reactive oxygen species are readily produced during auto-oxidation of Hb. On the other hand, Hb is known to have peroxidase-like activity monovalently oxidizing various peroxidase substrates. Thus, monovalently oxidized organic free radical species may be produced. This may relay the radical reactions leading to the production of reactive oxygen species such as superoxide. Such substrates possibly generating superoxide, include aromatic monoamines such as neurotransmitters and their precursors rich in neural a tissues. Based on our knowledge on the reactivity of haemoproteins against phenolics and aromatic monoamines, we proposed a hindered danger in use of Hb as a reperfusion agent. Clinical use of recently developing Hb-based blood substitutes must be reconsidered. © 2002 Elsevier Science Ltd

Abstract: Three Hyphodiscus species are described and illustrated: Hyphodiscus otanii sp. nov., Hyphodiscus hymeniophilus, and H. theiodeus, which is new to Japan. Culture studies revealed Phialophora-like anamorphs. Catenulifera gen. nov. is proposed for the anamorph of Hyphodiscus. The history of the genus is reviewed. Hyphodiscus can be delimited to members with gelatinized excipulum, Cistella-like hairs with more coarse granulation, small asci, ascospores with conspicuous globules, cylindrical, flexuous paraphyses, and a Catenulifera anamorph.

Abstract. Long-term changes in craniopharyngioma treated with radiation therapy (RT) were investigated by computed tomography (CT) and/or magnetic resonance (MR) imaging. Eight patients with craniopharyngioma were treated with incomplete resection or conservative surgical intervention followed by postoperative RT. The periods of tumor shrinkage were often long and varied (range: 6–68 months, mean: 29.1 months). Temporary enlargement of the solid component of a tumor usually occurs during RT and does not represent tumor progression. Cystic enlargement also occurs sometimes comparatively early after RT, and enlarged cysts often shrink with no treatment or with conservative treatment. These changes should be differentiated from tumor recurrence, with careful follow-up. After shrinkage, small solid or cystic nodules enhanced with contrast medium often remain. Long-term follow-up is necessary to differentiate uncontrolled tumors from controlled tumors with imaging modalities.

AbstractThe low calcium (Ca2+) dialysate have been developed to diminish the risk of hypercalcemia with the administration of active vitamin D and Ca2+ carbonate as phosphate binder. Today, increasing numbers of hemodialysis (HD) patients have been on the low Ca2+ dialysate (Ca2+ = 2.5 mEq/l). However, the clinical consequences of a negative calcium net-balance which may be induced by the use of low Ca dialysate are not well evaluated. In the present study, we explored the effects of low Ca2+ dialysate on the calcium balance and the PTH secretion. Eighty one chronic HD patients (male/female: 47/34; mean age: 60.2 ± 1.5 years; mean HD periods: 11.1 ± 0.8 years) who had been dialyzed with 3.0 mEq/l Ca2+ dialysate were studied. All patients were transferred to the low Ca dialysate, which actually brought about a negative net-balance in Ca (mean: –94.5 mg) and an increase in serum intact PTH levels (mean: +23.7%: p = 0.03) during a single HD session. However, no changes in serum ionized Ca2+ were found in spite of negative Ca2+ balance. One month after change to the low Ca2+ dialysate (total 12 sessions in each case), serum intact PTH levels increased significantly (186.7 ± 19.5 vs. 216.2 ± 21.9 pg/ml: p = 0.01) in spite of the fact that no changes were found in serum ionized Ca2+, Pi and Mg. This result indicates that the negative Ca2+ balance during low-Ca2+ hemodialysis-stimulated PTH secretion, which offset the decrease of serum Ca2+; a trade-off phenomenon between negative Ca balance and PTH. This suggests that low Ca2+ dialysate may exaggerate the progression of secondary hyperparathyroidism.Copyright © 2002 S. Karger AG, Basel

Technetium-99m-galactosyl human serum albumin (99mTc-GSA) is a radiopharmaceutical that binds to asialoglycoprotein receptors in the hepatocyte membrane; the level of 99mTc-GSA accumulation in the liver correlates well with the parameters of hepatic function tests. We hypothesized that the early blood kinetics of 99mTc-GSA could be described by a simple one-compartment model expressed as an offset a monoexponential function (y y0Ae−αt). The aims of this study were to assess the validity of this hypothesis using sequentially measured blood levels of 99mTc-GSA, which were approximated by a regression expression, and to analyse the regression equation. 99mTc-GSA levels were measured in blood samples collected from 30 patients with liver disease. From sequential changes in radioactivity up to 30 min after injection, a regression equation expressed as an offset a monoexponential function was calculated and evaluated by multiple correlation coefficients and absolute deviations ( error). The liver uptake rate 15 min after injection of 99mTc-GSA (liver uptake rate 15, UTR 15) was calculated from the regression equation in each case. The respective relationships of this parameter to the coefficient Aand gradient α were evaluated. In all cases, the measured values of radioactivity in blood samples could be fitted to an offset a monoexponential function. The validity of our hypothesis was supported by examining regression equations calculated from blood 99mTc-GSA levels. Analysis of the regression equations suggested a potential for a new index of hepatic functional reserve.

Abstract: We describe contrast enhancement of the cisternal portion of the abducens nerve and discuss its clinical significance. We examined 67 patients with ophthalmoplegia using contrast-enhanced 3-dimensional (3D) MRI with multiplanar reconstruction along the nerves and found 16 patients (ten men, six women), aged 10–73 years (mean 34.4 years), with contrast enhancement of the abducens nerve. Of the 36 patients who had an abducens palsy, 14 (39 %) showed contrast enhancement. In the 16 patients, 23 abducens nerves enhanced; 13 were symptomatic and 10 asymptomatic at the time. The causes were disseminated tumour (1), an inflammatory process (3), trauma (2), ischaemia (2) and autoimmune diseases (8), such as the Miller Fisher syndrome, acute ophthalmoparesis, polyneuropathy and multiple sclerosis. Abducens and/or oculomotor nerve enhancement was the only abnormality on MRI in the patients with traumatic or ischaemic neuropathy or autoimmune diseases. There were 14 patients who recovered fully within 1–6 months after treatment, and resolution of the enhancement correlated well with recovery.

Abstract: Background. Metastatic lung calcification (MLC) is an important complication in long-term dialysis patients, causing respiratory disturbance. We used single photon emission computed tomography (SPECT) in lung (L-SPECT) to detect the distribution of MLC. Methods. L-SPECT with 99mtechnetium hydroxymethylenediphosphonate was performed in 59 patients who had been on maintenance dialysis for more than 5 years. The accumulation count ratios were calculated from counts in the upper lung field (UL), lower lung field (LL), basal lung field along the diaphragm (BASE), and the cardiac pool (POOL). Distribution patterns of radionuclide were classified into four groups. Pulmonary function tests and blood examinations were performed. Results. The median values for UL/POOL, LL/POOL, and BASE/POOL were 1.71, 1.85, and 2.21, respectively. On the SPECT images, 28 patients had positive lung accumulation, versus 7 on the planar images. Patterns and frequency were: group I (none or slight accumulation), 32.2%; group II (accumulation only in the basal lung field), 33.8%; group III (accumulation in the whole lower lung or above), 18.6%; and group IV (diffuse accumulation extending to the whole lung field), 15.2%. Intact osteocalcin level was 6.3 ± 5.3 ng/ml in groups III + IV (large group) vs 94.2 ± 184.3 in groups I + II (small group) (P < 0.01). The number of patients with declining diffusion capacity was 20.0% in the large group, vs 5.1% in the small group. All patients in group IV had an intact parathyroid hormone (i-PTH) level of under 65 pg/ml. Conclusions. L-SPECT is very useful for the early detection of MLC, the extension of which may reduce diffusion capacity. Diffuse calcification over the whole lung is considered to be related to a low-turnover bone state concomitant with low PTH level.

Abstract: Because the transverse pontine fibres degenerate in some subtypes of spinocerebellar degeneration (SCD), demonstration of these fibres may be helpful for radiological diagnosis of SCD. Using multishot diffusion-weighted MRI, we attempted to find a way to show the transverse pontine fibres. We assessed the quality of demonstration of these fibres on DWI and of abnormal high signal in the pons and middle cerebellar peduncles on T2-weighted images. We examined evaluated 24 control subjects and 12 patients with SCD: two with sporadic olivopontocerebellar atrophy (OPCA), five with spinocerebellar ataxia type 1 (SCA1), two with SCA3, and three with SCA6. In all control subjects and patients with SCA6, we succeeded in demonstrating the transverse pontine fibres as clear low-signal bundles using DWI. In two patients with SCA3, these fibres were identified less distinctly. In contrast, in two patients with sporadic OPCA and in four of five patients with SCA1, the fibres were not identified. In both patients with sporadic OPCA, abnormal high-signal foci were seen in the base of the pons and middle cerebellar peduncles on T2-weighted images; no such foci were detected in any patient with SCA1, SCA3 or SCA6. DWI seems to be useful for demonstrating transverse pontine fibres. Abnormal high signal in the pons and middle cerebellar peduncles may provide a clue to differentiation of sporadic OPCA from other types of SCD.

Abstract: Background. Interleukin-1 (IL-1) has been reported to play a major role in the initiation and progression of several glomerulonephritis, and inhibition of IL-1 by the blockade of IL-1β-converting enzyme (ICE) has been suggested to be an ideal therapeutic strategy. Methods. To examine the effect of ICE inhibition on glomerulonephritis, we examined the susceptibility of ICE-deficient mice (ICE−/−) to anti-glomerular base-ment membrane antibody-induced glomerulonephritis (antiGBMGN), which has been previously reported to be mediated by IL-1β. Results. After the injection of antiGBM antibody to ICE−/− and wild type mice, albuminuria rose progressively and both groups of mice died within 7–9 days. Laboratory analysis of proteinuria, serum creatinine, and glomerular histology revealed no significant difference between the two groups. To pursue the mechanism of this result, bone marrow-derived monocyte/macrophage lineage cells (Mo/Mq cells) were established from both groups and the potency of IL-1β production in response to lipopolysaccharide was examined. An enzyme-linked immunosorbent assay (ELISA) of IL-1β revealed that, Mo/Mq cells from ICE−/− mice secreted IL-1β in response to lipopolysaccharide, although to a lesser extent than the Mo/Mq cells from ICE+/+ mice, suggesting that other protease(s) may process proIL-1β to generate the mature form. In fact, as was seen in the wild type mice, serum from antiGBM-injected ICE−/− mice contained IL-1β. Conclusions. These data suggest a limited effectiveness of ICE inhibition as a therapeutic strategy for glomerulonephritis.

In the development of the Drosophila central nervous system, some of the neuroblasts designated as neuroglioblasts generate both glia and neurons. Little is known about how neuroglioblasts produce these different cell types. NB6-4 in the thoracic segment (NB6-4T) is a neuroglioblast, although the corresponding cell in the abdominal segment (NB6-4A) produces only glia. Here, we describe the cell divisions in the NB6-4T lineage, following changes in cell number and cell arrangement. We also examined successive changes in the expression of glial cells missing (gcm) mRNA and protein, activity of which is known to direct glial fate from the neuronal default state. The first cell division of NB6-4T occurred in the medial-lateral orientation, and was found to bifurcate the glial and neuronal lineage. After division, the medial daughter cell expressed GCM protein to produce three glial cells, while the lateral daughter cell with no GCM expression produced ganglion mother cells, secondary precursors of neurons. Although gcm mRNA was present evenly in the cytoplasm of NB6-4T before the first cell division, it became detected asymmetrically in the cell during mitosis and eventually only in the medial daughter cell. In contrast, NB6-4A showed a symmetrical distribution of gcm mRNA and GCM protein through division. Our observations suggest that mechanisms regulating gcm mRNA expression and its translation play an important role in glial and neuronal lineage bifurcation that results from asymmetric cell division.

Although many pathological changes in the internal capsule may lead to neurological deficits, we often encounter ill-defined focal low attenuation in the posterior limb of the internal capsule (PIC) on CT in patients with no neurological disturbance. Brain CT studies of 141 patients without neurological deficits were reviewed to investigate the position of the focal low attenuation by analysis of a profile density curve. Nine patients with lacunar infarcts only within the posterior internal capsule were also studied. The focal low attenuation areas were ill-defined and bilaterally symmetrical, without mass effect. They were seen consistently within the posterior limb of the posterior internal capsule. Correlation between the distribution of these foci and the position of lacunar infarcts in the posterior internal capsule in nine patients with neurological deficits suggests that they may be related to the corticospinal tract.

Although many pathological changes in the internal capsule may lead to neurological deficits, we often encounter ill-defined focal low attenuation in the posterior limb of the internal capsule (PIC) on CT in patients with no neurological disturbance. Brain CT studies of 141 patients without neurological deficites were reviewed to investigate the position of the focal low attenuation by analysis of a profile density curve. Nine patients with lacunar infarcts only within the posterior internal capsule were also studied. The focal low attenuation areas were ill-defined and bilaterally symmetrical, without mass effect. They were seen consistently within the posterior limb of the posterior internal capsule. Correlation between the distribution of these foci and the position of lacunar infarcts in the posterior internal capsule in nine patients with neurological deficits suggests that they may be related to the corticospinal tract.

Bone matrix gelatin, prepared by sequential chemical treatment including decalcification with 0.6 N hydrochloric acid [9], was used as an alloimplant for the treatment of benign bone tumours, tumorous conditions of bone, acetabular dysplasia, delayed union, traumatic bone defects and other disorders. The bone matrix gelatin implanted into bone defects was incorporated successfully in 98% of implantations, excluding cases of infection, tumour recurrence and recurrence of tumorous conditions. The material was also implanted into ten bone sites as an onlay but in five it was resorbed without new bone formation. The incorporation of the bone matrix gelatin into the recipient bed was completed from 6 to 33 months (average 14.9 months) after implantation. Wound infection complicated 5 of 165 implantations (3%) in previously uninfected sites. Low grade fever persisting after the tenth post-operative day (a probable sign of immunological reaction) occurred in 4 of 160 implantations (2.5%), excluding cases of infection. Alloimplants of bone matrix gelatin are thus effective in the treatment of bone defects. The risk of complication such as rejection or infection is low.

The Drosophila visual mutant, carrying the retinal degeneration A gene (rdgA), has photoreceptor cells that degenerate within a week after eclosion. Morphological studies suggested that this mutant harbors abnormalities in membrane turnover of the photoreceptor cells. Biochemically, the rdgA mutant lacks an eye-specific and membrane-associated diacylglycerol kinase (DGK; EC 2.7.1.107) activity in a gene-dosage-dependent manner, suggesting that rdgA gene encodes a DGK. We report the molecular cloning and characterization of a DGK gene, which maps to the rdgA locus. This gene, designated as DGK2, has a single open reading frame that encodes 1454 amino acids. Like porcine DGK, DGK2 has two cysteine-rich zinc-finger motifs as well as a DGK catalytic domain. The DGK2 protein contains four ankyrin-like repeats at the C-terminal region, suggesting that DGK2 is likely anchored to the membrane or cytoskeleton. Northern blot analysis and tissue in situ hybridization to adult sections revealed that DGK2 is expressed exclusively in the adult retina and that the amount of its mRNA is reduced in some of the rdgA mutant alleles. Furthermore, in two rdgA alleles, rdgA1 and rdgA2, nonsense and missense mutations occur within their DGK2 gene, respectively. Thus, we conclude that rdgA encodes an eye-specific DGK, the absence of which leads to rhabdomere degeneration due to defective phospholipid turnover.

Thirty-seven femoral heads in idiopathic avascular necrosis of adults and fifty-seven biopsy specimens of the capital nucleus of Perthes' disease were studied histologically. Although the early pathological changes in these two diseases are very similar, in the intermediate and late (advanced) stages they gradually take differing courses. Although the initiating causes in both diseases are still obscure both are probably produced by repeated episodes of infarction of the femoral head.

Abstract: We performed a histopathological study of two human brains to look at dilated Virchow-Robin (V-R) spaces in the anterior perforated substance and putamen. We measured the diameter of 74 arteries in 54 dilated V-R spaces. In 28 patients without neurological deficits we ascertained the characteristic location of dilated V-R spaces in the anterior perforated substance and basal ganglia on MRI, measuring the distance from 64 foci of cerebrospinal fluid signal intensity to the centre of the mamillary body on 1 mm thick images. In the histopathological study, the mean diameter of the arteries was 39.0 ± 36.0 μm. Dilatation of the V-R space was observed from the end of the indentation of the pial membrane towards the brain surface along the perforating artery. In the MR images, the mean distance from the dilated V-R space to the mamillary body was 10.0 ± 4.5 mm. The V-R space was confined to a fixed level in the lower part of the basal ganglia, and not found near the brain surface.

Isolation of beta-linkage-containing side chain oligosaccharides from the mannan of Candida guilliermondii IFO 10279 strain has been conducted by acetolysis under mild conditions. A structural study of these oligosaccharides by one- and two-dimensional NMR and methylation analyses indicated the presence of extended oligosaccharide side chains with two consecutive beta-1,2-linked mannose units at the nonreducing terminal of alpha-linked oligosaccharides. The linkage sequence present in this mannan, Man beta 1-->2Man alpha 1-->3Man alpha-->, has also been found in the mannan of Saccharomyces kluyveri but not in the mannan of Candida species. Furthermore, these oligosaccharides are branched at position 6 of the 3-O-substituted mannose units as follows. (Carbohydrate sequence in text) Structure 1 and (Carbohydrate sequence in text) Structure 2 The H-1 signals of the mannose units substituted by a 3,6-di-O-substituted unit showed a significant upfield shift (delta delta = 0.04-0.08 ppm) due to a steric effect. The inhibition of an enzyme-linked immunosorbent assay between the mannan of C. guilliermondii and factor 9 serum with oligosaccharides obtained from several mannans indicated that only the oligosaccharides with the above structure were active, suggesting that these correspond to the epitope of antigenic factor 9.

Abstract:  The glial cells missing (gcm) gene encodes an essential transcription factor that converts neuronal precursor cells to glial fate in the Drosophila nervous system. In this study, we tested effects of gcm ectopic expression on fate of non-neural cells. When gcm expression was continuously induced in epidermal cells from around stage 9, these cells started to exhibit mesenchymal cell morphology at stage 13, which was preceded by the onset of expression of Repo, a glial marker. The morphological change was coincident with loss of expression of an epidermal cell-adhesion molecule. In addition to the epidermis, fate of mesodermal cells was also affected by gcm ectopic expression. These findings suggest that gcm can convert gene expression and cell morphology even outside the neuroectoderm.

Nucleolin is a major nucleolar phosphoprotein and is presumably involved in rDNA transcription and ribosome biosynthesis. This protein is known to be very labile and to be cleaved by endogenous proteases into many small peptides. We found that, when rat liver nucleolar suspension (Nu-1) or nucleolin-rich extract (Nu-2) was incubated under conventional conditions, polyamines and histones interacted with the nucleolin to lead to its preferential degradation to 60 kDa phosphopeptide (p60). The peptide p60 was identified as a peptide containing the N-terminal half of the nucleolin molecule, as judged from peptide-map analysis. Whereas spermine binding to the purified nucleolin was decreased by KCl concentrations above 50 mM, histones (H1, H2B and H3) were able to bind to the nucleolin in the presence of up to 300 mM KCl. A distinct difference between H1 and other histones was found in that H1 could produce p60 from nucleolin in both Nu-1 and Nu-2, whereas H2B and H3 stimulated the degradation of nucleolin to p60 only when Nu-2 was used for the source of nucleolin. A possible relationship between p60 formation and rRNA synthesis is discussed, but its exact role remains to be studied.

In order to minimize the transmission lags associated with the pneumatic instrumentation, the Transmission Lag Compensator (TLC) was developed. The construction and features of the TLC which is based on the phase lead compensation are described. The effects of applying the TLCs are investigated by experiments and simulations. Experiments with practical plants using the TLCs show the improvement of the control performance over the system without the TLC. The effects of applying the TLCs are also obtained quantitatively from the control performance simulations.

The primary and gene structures and tissue distribution of porcine SPAI-2, a protein that belongs to the WAP protein superfamily and has a sodium-potassium ATPase inhibitory activity, were determined by molecular cloning and Northern analysis. A full-length cDNA clone was isolated from a porcine duodenum cDNA library. The cDNA insert encoded a polypeptide of 187 amino acids, which is composed of three domains: a hydrophobic presequence of 21 amino acids, a prosegment of 105 amino acids ending with Asp126, and the mature SPAI-2 sequence of 61 amino acids beginning with Pro127. The prosegment contained 16 repeats of a hexapeptide that is highly homologous to the repetitive sequence found in the transglutaminase domain of the human elafin, an elastase-specific inhibitor that also belongs to the WAP superfamily. The repetitive sequence was demonstrated to be a good substrate of transglutaminase using a recombinant preparation produced in Escherichia coli. A porcine genomic library was then screened for the SPAI gene. Characterization and sequencing of positive clones indicated that the gene is similar to the elafin gene, having 3 exons encoding the 5'-untranslated region and signal sequence, proSPAI, and 3'-untranslated region, respectively. Northern blot analysis revealed intestine-specific expression of SPAI mRNA; the message was especially abundant in the small intestine. ProSPAI was also found in the circulation. The similarity of proSPAI to elafin in the domain structure, the acid-labile nature of the cleavage site (Asp126-Pro127), and the fact that the major form of SPAI in the plasma is proSPAI strongly suggest that proSPAI is not the precursor but rather it is the native form of SPAI. Like elafin, therefore, SPAI appears to be a new type of biologically active substance with a transglutaminase substrate domain that acts as an anchoring sequence.

Abstract: MRI was obtained in eight normal volunteers and seven patients with brain oedema around the trigone. In addition to the conventional sequences, diffusion-weighted and intravoxel-incoherent-motion images using motion-proving anteroposterior and/or lateral direction gradients were obtained to show the white matter pathways better. Coronal proton-density-weighted images showed three thin relatively high-intensity layers in addition to the tapetum and the internal and external sagittal strata. Although they have not been confirmed anatomically, the thin layer between the internal and the external sagittal strata was corroborated by diffusion-weighted and intravoxel-incoherent-motion images, and by characteristics of the spread of oedema into the sagittal stratum. We propose that this layer be named the central sagittal lamina. The other two layers medial and lateral to the sagittal stratum were outside, but in contact with the medial and lateral parts of the sagittal stratum, respectively. We provisionally named them medial and lateral sagittal laminae; they were not evident on any other images. The low-intensity layer on T2-weighting was the internal sagittal stratum. The optic radiation, comprising the external sagittal stratum, appeared as an intermediate to slightly high-intensity layer on T2-weighted images and a low-intensity layer on T1-weighted images as did the corticospinal tract in the posterior internal capsule.

We assessed the results of Chiari pelvic osteotomy in 64 hips with early osteoarthritis in terms of acetabular labral detachment detected pre-operatively by arthrography. At an average follow-up of four years, assessment by the Merle d'Aubigne score showed 83% excellent or good results. These satisfactory results were achieved in only half the cases with a detached labrum, but in nearly all cases with normal or torn acetabular labra. Other factors such as the acetabular index, the level and angle of osteotomy, and the displacement following osteotomy did not affect the results. Chiari pelvic osteotomy is a worthwhile procedure for early osteoarthritis in selected cases, but a detached acetabular labrum increases the risk of clinical failure.

The geometry of hydrogen donor molecules bound to horseradish peroxidase was investigated using nuclear magnetic resonance techniques. Between resorcinol and 2-methoxy-4-methylphenol which showed different optical difference spectra, little difference was observed in the orientation of the molecules bound to horseradish peroxidase: the minimal distances between the enzyme iron and the protons of the phenol rings are in the range of 8.4-11.0 A. This situation was not greatly different for the third compound studied in this paper, benzhydroxamic acid, providing evidence against the view that its side chain coordinates to the heme iron. Furthermore, it was found that transferred nuclear Overhauser effect for the signals of these compounds was observable only when the heme peripheral 8-methyl proton signal was irradiated. These results, together with a hypothetical model of the enzyme structure obtained by computer-aided simulation procedures, suggest that the binding of these donor molecules and competitive inhibitors occur in the vicinity of the heme peripheral 8-methyl group, with hydrophobic interactions probably with Tyr-185 and with hydrogen bond with adjacent amino acid residues such as Arg-183.

The effects of testosterone (in vivo) and polyamines (in vitro) on the phosphorylation of nucleolar proteins of rat ventral prostates were studied. Phosphorylation of nucleolar proteins was accomplished by incubation of isolated nucleoli with [gamma-32P]ATP at 37 degrees C for 10 min followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Of several nucleolar phosphoproteins observed in ventral prostates of castrated rats, the incorporation of 32P into 110-kDa protein was remarkably augmented by the testosterone treatment. The stimulation became evident as early as 4 h after the injection of the hormones, reaching 3-4-fold of the control level and was efficiently prevented by cycloheximide injection 3 h before killing. 5 alpha-Dihydrotestosterone gave similar results to testosterone, but estradiol-17 beta failed to stimulate the phosphorylation of 110-kDa protein. Polyamines and cyclic nucleotides did not affect the phosphorylation, but, when phenylmethanesulfonyl fluoride was omitted from the standard medium, spermine and spermidine showed a distinct effect: 110-kDa phosphoprotein was completely abolished with a concomitant increase of 59-kDa phosphoprotein in both cases of castrated and testosterone-primed rats. The effect of polyamines seems to be due to the stimulation of degradation of the protein which is presumably catalyzed by a serine protease.

Binding of an iodide ion to horseradish peroxidase was studied by following the hyperfine-shifted proton nuclear magnetic resonance signals of the enzyme. For the enzyme in an iodide-free solution, the spectra of hyperfine-shifted methyl region were only slightly affected by varying pH. In the presence of iodide (200 mM), however, both chemical shifts and line widths of the heme peripheral 1- and 8-methyl proton signals were markedly affected by the pH change from 7 to 4 and broadened at pH 4. From the change in peak heights of these signals at various concentrations of iodide, the dissociation constant of the iodide to the enzyme was calculated to be about 100 mM at pH 4.0. The peak derived from the proximal histidyl imidazole N epsilon-H proton was not perturbed by the addition of 200 mM iodide at pH 4.0 and 7.1. The rate of oxidation of iodide with hydrogen peroxide catalyzed by the enzyme was increased with decreasing pH, indicating the participation of an ionizable group with the pKa value of 4.0. Optical difference spectrum studies showed that iodide exerts no effect both at pH 4.0 and 7.4 on the binding affinity of resorcinol which is associated with the enzyme in the vicinity of the heme peripheral 8-CH3 group. These results suggest that an iodide ion binds to the enzyme at almost equal distance from the heme peripheral 1- and 8-methyl groups at the distal side of the heme and that the interaction becomes stronger in acidic medium with protonation of the ionizable group with the pKa value of 4.0.

In vivo effects of dexamethasone and cycloheximide on the phosphorylation of nucleolar proteins and the activities of nucleolar protein kinases of rat livers were studied. Phosphorylation of nucleolar proteins was accomplished by incubation of isolated nucleoli with [gamma-32P]ATP at 37 degrees C for 10 min followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Of several nucleolar phosphoproteins observed in the liver of adrenalectomized rats, the incorporation of 32P into nucleolar 110-kDa proteins was rapidly increased, reaching about 1.8-fold of the adrenalectomized level at 12 h after administration of dexamethasone. In addition, the activities of liver nucleolar protein kinases which were measured using casein or phosvitin as substrates were stimulated similarly by the administration of dexamethasone. It was found by DEAE-Sephadex column chromatography that nucleolar protein kinases were separated into NI and NII, the activities being increased about 35 and 80% by dexamethasone treatment (12 h), respectively, and that the enhancements were completely abolished by 1-h treatment of cycloheximide. The injection of cycloheximide also suppressed the hormone-induced enhancement of 32P incorporation into 110-kDa proteins but did not reduce the amount of the proteins at least until 2 h. Evidence was obtained indicating that the phosphorylation of the nucleolar 110-kDa proteins is accomplished primarily by protein kinase NII. These results suggest that continuous synthesis of protein(s) is necessary for glucocorticoid-induced enhancement of phosphorylation of 110-kDa proteins of liver nucleoli and that the nucleolar protein kinase NII is involved in the glucocorticoid-induced short-lived protein(s).

In order to examine the relationship between RNA polymerase I and ornithine decarboxylase (ODC), three lines of experiments were performed, with the following results. The glucocorticoid-induced increase of RNA polymerase I in rat liver nuclei was not abolished by administration of inhibitors of ODC synthesis and activity, namely 1,3-diaminopropane and 2-difluoromethylornithine respectively. Anti-ODC antibody did not cross-react with RNA polymerase I solubilized from rat liver nucleoli, indicating the absence of a common protein sequence in these enzymes. The ODC preparation which was treated with transglutaminase in the presence of putrescine could not stimulate the activity of RNA polymerase I in nuclei of liver and prostate. All these results suggest that the increases in ODC protein or activity are not a prerequisite to the increase in RNA polymerase I after hormonal or physiological stimuli, but rather that the increases in both enzymes are separate responses to the primary stimuli.

The activity of the template-engaged form of RNA polymerase I from livers of adrenalectomized rats was about 50-60% of that of normal control rats, and increased about 2-fold at 6 h after the administration of dexamethasone. However, no change was found in the activity of the ‘free’ form of RNA polymerase I or the template-engaged form of RNA polymerase II. Immunochemical studies using guinea-pig anti-(RNA polymerase I) serum disclosed that the total number of RNA polymerase I molecules did not vary during the treatment with dexamethasone. Cycloheximide caused a rapid decrease in the template-engaged form of RNA polymerase I activity in normal rats and in dexamethasone-treated (6 h) adrenalectomized rats, to the value in adrenalectomized rats, but affected it only slightly in adrenalectomized rats. The elongation rate of rRNA-precursor synthesis in liver nuclei was not affected by a change in the concentration of circulating dexamethasone. From these results, it is concluded that about half the rRNA-precursor synthesis in rat liver is regulated by glucocorticoids, probably through the synthesis of short-lived protein(s) which may play a role in conversion of the ‘dormant’ form of RNA polymerase I into the ‘engaged’ form.

The site and characteristics of iodide binding to Arthromyces ramosus peroxidase were examined by x-ray crystallographic analysis, 1H and 127I NMR, and kinetic studies. X-ray analysis of an A. ramosus peroxidase crystal soaked in a KI solution at pH 5.5 showed that a single iodide ion is located at the entrance of the access channel to the distal side of the heme and lies between the two peptide segments, Phe90-Pro91-Ala92 and Ser151-Leu152-Ile153, 12.8 A from the heme iron. The distances between the iodide ion and heme peripheral methyl groups were all more than 10 A. The findings agree with the results obtained with 1H NMR in which the chemical shift and intensity of the methyl groups in the hyperfine shift region of A. ramosus peroxidase were hardly affected by the addition of iodide, unlike the case of horseradish peroxidase. Moreover, 127I NMR and steady-state kinetics showed that the binding of iodide depends on protonation of an amino acid residue with a pKa of about 5.3, which presumably is the distal histidine (His56), 7.8 A away from the iodide ion. The mechanism of electron transfer from the iodide ion to the heme iron is discussed on the basis of these findings.

We have isolated a Drosophila melanogaster diacylglycerol kinase (DGK, EC 2.7.1.107) homologue by using a porcine DGK cDNA probe and we have characterized its structure and expression. The DGK cDNA has a single open reading frame that encodes 791 amino acids. The Drosophila and porcine DGKs share a similar carboxyl-terminal region, a putative catalytic domain, which is divided into two separate domains in Drosophila. The DGK gene was mapped to the cytogenetic position 43F1, and its DGK mRNA is abundant both in embryo and in adult fly. By in situ hybridization to sections of adult flies, we demonstrated that the mRNA is present predominantly in the nervous system and muscles, including compound eyes, brain cortex, fibrillar muscle, and tubular muscle. In a 10- to 11-hr embryo, the DGK gene is expressed abundantly in a limited number of cells in the procephalic region and in the ventral nerve cord. The pattern of temporal and spatial expression suggests that the DGK protein has an important function in the adult nervous system and muscle and during the development of the embryonic nervous system.

The coupling of iodotyrosine residues of thyroglobulin (Tg) catalysed by thyroid peroxidase (TPO) has scarcely been studied with respect to the TPO of abnormal human thyroid glands. The present paper proposes a rapid and convenient assay method applicable for determining the coupling activity of a sample of less than 500 mg from each patient's thyroid. The main characteristics of the method are as follows: (i) mitochondrial/microsomal fractions of thyroid glands were treated with sodium cholate plus trypsin, and the supernatants obtained by ultracentrifugation were directly used for the assay of coupling and peroxidase activity of TPO; (ii) the formation of iodotyrosine residues catalysed by TPO was performed by using chemically iodinated Graves'-disease Tg containing 41 iodine atoms per molecule and with a high iodotyrosine and a low iodothyronine content; (iii) newly synthesized iodothyronine residues (thyroxine, 3,5,3′-tri-iodothyronine, and 3,3′,5′-tri-iodothyronine) were analysed by h.p.l.c. after hydrolysis of Tg with proteinases and extraction of iodothyronines with ethyl acetate.

Selective percutaneous ethanol injection therapy (PEIT) has been used to control parathyroid function in patients with secondary hyperparathyroidism (2HPT) when one or more parathyroid gland (PTG) progresses to the nodular hyperplasia stage. However, PEIT can have adverse side effects, such as nerve paralysis and adhesion, because the ethanol is destructive. Intraparathyroid injection of a vitamin D analogue has been designed as a treatment to control parathyroid function without destruction of the PTG or causing adhesions to the surrounding tissue, and the present study aimed to verify the effect of percutaneous maxacalcitol (22-oxacalcitriol) as the vitamin D analogue. The study group comprised two haemodialysis patients who needed parathyroidectomy for uncontrolled 2HPT with a maximal PTG diameter >20 mm. The treatment began with an ultrasonographically guided injection of 10 µg of maxacalcitol solution into the largest PTG and, 1 week later, parathyroidectomy was performed to examine the effect of the maxacalcitol injection both macroscopically and microscopically. The injected glands were swollen and inflamed, and adhesions made it difficult to remove them. There was macroscopic and microscopic evidence of haemorrhagic necrosis and adhesions to the surrounding tissue. Direct vitamin D analogue injection should not be performed as a primary treatment option because the adverse side effects are not overcome by this technique.

Selective percutaneous ethanol injection therapy (PEIT) has been used to control parathyroid function in patients with secondary hyperparathyroidism (2HPT) when one or more parathyroid gland (PTG) progresses to the nodular hyperplasia stage. However, PEIT can have adverse side effects, such as nerve paralysis and adhesion, because the ethanol is destructive. Intraparathyroid injection of a vitamin D analogue has been designed as a treatment to control parathyroid function without destruction of the PTG or causing adhesions to the surrounding tissue, and the present study aimed to verify the effect of percutaneous maxacalcitol (22-oxacalcitriol) as the vitamin D analogue. The study group comprised two haemodialysis patients who needed parathyroidectomy for uncontrolled 2HPT with a maximal PTG diameter >20 mm. The treatment began with an ultrasonographically guided injection of 10 µg of maxacalcitol solution into the largest PTG and, 1 week later, parathyroidectomy was performed to examine the effect of the maxacalcitol injection both macroscopically and microscopically. The injected glands were swollen and inflamed, and adhesions made it difficult to remove them. There was macroscopic and microscopic evidence of haemorrhagic necrosis and adhesions to the surrounding tissue. Direct vitamin D analogue injection should not be performed as a primary treatment option because the adverse side effects are not overcome by this technique.

AbstractBackground/Aims: Familial juvenile gouty nephropathy (FJGN, MIM 162000) is an autosomal-dominant renal disease characterized by underexcretion-type hyperuricemia, gouty arthritis, and progressive renal disease at younger ages. We analyzed the localization of the responsible gene for FJGN concerning the chromosomal region of 16p12 using parametric linkage analysis in our FJGN. Methods: The affected members of this family were accompanied with polyuria due to nephrogenic diabetes insipidus and without hypertension. Fifteen samples were collected from 9 affected and 6 nonaffected members of the family. By using microsatellite markers mainly focused on the short arm of chromosome 16, two point and multipoint linkage analyses were carried out. Results: All of the 2-point logarithm of odds (LOD) scores were typically negative and all of the multipoint LOD scores were less than –3.0 in our FJGN family. Conclusion: The results suggested that the localization of the responsible gene to 16p12 can be excluded in our FJGN family. This finding means that the responsible gene for FJGN is not common.Copyright © 2002 S. Karger AG, Basel