Members of the transforming growth factor-β (TGF-β) superfamily participate in numerous biological phenomena in multiple tissues, including in cell proliferation, differentiation, and migration. TGF-β superfamily proteins therefore have prominent roles in wound healing, fibrosis, bone formation, and carcinogenesis. However, the molecular mechanisms regulating these signaling pathways are not fully understood. Here, we describe the regulation of bone morphogenic protein (BMP) signaling by Bat3 (also known as Scythe or BAG6). Bat3 overexpression in murine cell lines suppresses the activity of the Id1 promoter normally induced by BMP signaling. Conversely, Bat3 inactivation enhances the induction of direct BMP target genes, such as Id1, Smad6, and Smad7. Consequently, Bat3 deficiency accelerates the differentiation of primary osteoblasts into bone, with a concomitant increase in the bone differentiation markers Runx2, Osterix, and alkaline phosphatase. Using biochemical and cell biological analyses, we show that Bat3 inactivation sustains the C-terminal phosphorylation and nuclear localization of Smad1, 5, and 8 (Smad1/5/8), thereby enhancing biological responses to BMP treatment. At the mechanistic level, we show that Bat3 interacts with the nuclear phosphatase small C-terminal domain phosphatase (SCP) 2, which terminates BMP signaling by dephosphorylating Smad1/5/8. Notably, Bat3 enhances SCP2–Smad1 interaction only when the BMP signaling pathway is activated. Our results demonstrate that Bat3 is an important regulator of BMP signaling that functions by modulating SCP2–Smad interaction.

Human herpesvirus 6 (HHV-6)-associated encephalitis or pneumonitis has been reported in immunocompetent and immunosuppressed individuals. Several MRI studies in patients with HHV-6-associated encephalitis have been presented. However, to the best of our knowledge, no studies describing thin-section CT imaging in patients with HHV-6-associated pneumonitis have been reported. Here we describe a case of HHV-6-associated encephalitis and pneumonitis that developed after bone marrow transplantation. Thin-section CT images of the chest revealed ground-glass attenuation, consolidation and centrilobular nodules in both lungs.

Summary

We introduced our grading system that enables us to objectively evaluate the degree of technical difficulty of aneurysm treatment modalities, i.e. neck clipping and coil embolization. The characteristics of our grading system were that the difficulty of each treatment was indicated on a ten point scale obtained by adding the scores for various technical factors. We studied annual change of treatments selected for ruptured aneurysms and the treatment results at our institute. In the earlier half of the study period, neck clipping was more frequently selected despite the fact that the difficulty score of coil embolization was lower than that of neck clipping. However, in the later half, the treatment modality was selected in accordance with the difficulty score in most of the cases. As a result, there was a tendency for the proportion of mRS 0 to increase and that of mRS 6 to decrease as the years passed. Our grading system may be useful in objectively selecting a more appropriate treatment, and further improve treatment results.

Objective: To investigate whether the myocardial performance index (MPI) can predict left ventricular functional outcome in patients with early recanalisation after anterior acute myocardial infarction (MI) and to determine when the index should be measured.

Design: MPI was measured serially by two dimensional Doppler echocardiography after successful percutaneous coronary intervention (PCI). Left ventricular function was evaluated by echocardiography and left ventriculography. To assess coronary microvascular damage, the coronary flow velocity pattern was measured immediately after PCI with a Doppler guidewire.

Setting: Hiroshima City Asa Hospital.

Patients: 32 consecutive patients with their first anterior acute MI who had complete occlusion of left anterior descending coronary artery.

Interventions: Successful PCI within six hours of symptom onset.

Main outcome measures: Left ventricular anterior wall motion score index (A-WMSI), left ventricular end diastolic pressure (LVEDP), left ventricular ejection fraction (LVEF), and left ventricular end diastolic volume (LVEDV).

Results: There was a significant negative correlation between MPI on day 2 and the coronary diastolic deceleration time (r  =  −0.66, p < 0.002), as well as a significant positive correlation with the coronary diastolic deceleration rate (r  =  0.74, p < 0.0001). MPI on day 2 was significantly correlated with the short and long term changes of A-WMSI and with the short term changes of LVEDP. Furthermore, MPI on day 2 was significantly correlated with the short and long term changes of LVEF (r  =  −0.52, p < 0.003, and r  =  −0.64, p < 0.0008, respectively) and of LVEDV (r  =  0.51, p < 0.003, and r  =  0.41, p < 0.05, respectively).

Conclusions: Doppler derived MPI on day 2, representative of the early coronary microvascular state, can predict the left ventricular functional outcome after early successful recanalisation of a patient’s first anterior acute MI.

Objective: To test the hypothesis that the power of the received signal of harmonic power Doppler imaging (HPDI) is proportional to the bubble concentration under conditions of constant applied acoustic pressure, and to determine whether a new quantitative method can overcome the acoustic field inhomogeneity during myocardial contrast echocardiography (MCE) and identify perfusion abnormalities caused by myocardial infarction.

Methods: The relation between Levovist concentration and contrast signal intensity (CI) of HPDI was investigated in vitro under conditions of constant acoustic pressure. MCE was performed during continuous infusion of Levovist with intermittent HPDI every sixth cardiac cycle in 11 healthy subjects and 25 patients with previous myocardial infarction. In the apical views myocardial CI (CImyo) was quantified in five myocardial segments. The CI from the left ventricular blood pool adjacent to the segment was also measured in dB and subtracted from the CImyo (relative CI (RelCI)).

Results: CI had a logarithmic correlation and the calculated signal power a strong linear correlation with Levovist concentration in vitro. Thus, a difference in CI of X dB indicates a microbubble concentration ratio of 10X/10. In normal control subjects, CImyo differed between the five segments (p < 0.0001), with a lower CImyo in deeper segments. However, RelCI did not differ significantly between segments (p  =  0.083). RelCI was lower (p < 0.0001) in the 39 infarct segments (mean (SD) −18.6 (2.8) dB) than in the 55 normal segments (mean (SD) −15.1 (1.6) dB). RelCI differed more than CImyo between groups.

Conclusions: The new quantitative method described can overcome the acoustic field inhomogeneity in evaluation of myocardial perfusion during MCE. RelCI represents the ratio of myocardium to blood microbubble concentrations and may correctly reflect myocardial blood volume fraction.

Summary

Mutations in the genes for nuclear envelope proteins of emerin (EMD) and lamin A/C (LMNA) are known to cause Emery-Dreifuss muscular dystrophy (EDMD) and limb girdle muscular dystrophy (LGMD). We compared clinical features of the muscular dystrophy patients associated with mutations in EMD (emerinopathy) and LMNA (laminopathy) in our series. The incidence of laminopathy was slightly higher than that of emerinopathy. The age at onset of the disease in emerinopathy was variable and significantly older than in laminopathy. The initial symptom of emerinopathy was also variable, whereas nearly all laminopathy patients presented initially with muscle weakness. Calf hypertrophy was often seen in laminopathy, underscoring the importance of mutation screening for LMNA in childhood muscular dystrophy with calf hypertrophy. The clinical spectrum of emerinopathy is actually wider than previously known including EDMD, LGMD, conduction defects with minimal muscle/joint involvement, and their intermittent forms. Pathologically, no marked difference was observed between emerinopathy and laminopathy. Increased number and variation in size of myonuclei were detected. More precise observations using electron microscopy is warranted to characterize the detailed nuclear changes in nuclear envelopathy.

Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

Summary

This study evaluated: 1) the effect of recanalization on changing clinical outcome, 2) the relationship between dose of Urokinase (UK) and incidence of recanalization and intracranial haemorrhage, and 3) the efficacy and feasibility of balloon disruption (BD) in the treatment of acute cerebral embolism.

Sixty-one patients with acute embolism of the major cerebral arteries treated by endovascular approaches over the past nine years were retrospectively evaluated. Among them, 30 cases were treated by BD alone or in conjunction with intra-arterial fibrinolysis in the last five years. The other 31 cases, mostly treated in the first four years, were treated with intra-arterial fibrinolysis alone and were used as controls to evaluate the efficacy of BD. Control angiography was performed just after the reperfusion procedure to evaluate the degree of recanalization. Angiographic responses were graded using modified Thrombolysis in Myocardial Infarction (TIMI) criteria. Clinical outcome was evaluated using modified Rankin Scale (mRS) score at the time of discharge.

Thirty-six of the 61 patients (59.0%) achieved high-grade recanalization (TIMI grade 3). Significantly more patients attained favorable outcome (mRS score 0-1) in the high-grade recanalization group than the low-grade recanalization group (41.7% vs. 16.0%, p< 0.05). Concerning patients treated with BD, significantly more patients attained good recanalization and significantly more patients were ambulatory (mRS score 0-3) than those treated with intra-arterial fibrinolysis alone (76.7% vs. 41.9%, p<0.01; 70.0% vs. 41.9%, p< 0.05, respectively).A significantly lower dose of UK was used, and relatively less intracranial haemorrhage was seen in patients treated with BD than those treated with intra-arterial fibrinolysis (194,000 ± 191,000 units vs. 388,000 ± 231,000 units, p=0.001; 16.7% vs. 38.7%, p = 0.055, respectively). Concerning morbidity and mortality of BD, there was one death caused by dissection of the M2 portion of the middle cerebral artery (MCA) that happened during BD on a distally migrated embolus.

Although no conclusions can be drawn from our study, a favorable outcome for acute embolism of the major cerebral arteries is expected by attaining good recanalization. In addition, BD is an effective technique that can achieve high-grade recanalization alone, or reducing the dose of fibrinolytic agent.

We conducted a phase II trial of triplet chemotherapy consisting of vinorelbine, gemcitabine, and cisplatin in patients with advanced non-small cell lung cancer to assess its efficacy and toxicity. Thirty-three patients with chemotherapy-naïve stage IIIB disease (n=8), stage IV disease (n=23), or recurrence after surgical resection (n=2) were given intravenous infusions of vinorelbine 25 mg m−2, gemcitabine 1000 mg m−2, and cisplatin 40 mg m−2 on days 1 and 8 at 3-week intervals. There were 16 partial responses, and the objective response rate was 48% (95% confidence interval: 31–66%). The median survival time was 13.5 months (95% confidence interval: 10.6–16.4 months), and the one-year survival rate was 61%. Grade 4 haematologic toxicity consisted of neutropenia in 72% of patients, and febrile neutropenia occurred in 42% of the patients. There was one toxic death, and it was attributed to neutropenic fever and haemoptysis. Autopsy revealed diffuse pulmonary haemorrhage secondary to bacterial abscesses and vasculitis in both lungs. The common nonhaematologic toxicities included grade 2–3 nausea (39%) and vomiting (18%). Triplet chemotherapy containing vinorelbine, gemcitabine, and cisplatin is effective in the treatment of chemo-näive patients with advanced non-small cell lung cancer, but produces unacceptable frequent febrile neutropenia.

British Journal of Cancer (2002) 87, 1360–1364. doi:10.1038/sj.bjc.6600658 www.bjcancer.com

© 2002 Cancer Research UK

We assessed the requirement of the host cytoskeleton for the intracytosolic transport of incoming human cytomegalovirus (HCMV) capsids. Treatments with microtubule (MT)-depolymerizing drugs nocodazole and colchicine led to a drastic decrease in levels of IE1 antigen, whereas cytochalasin B had no effect on the level of IE1 as determined by Western blot analyses. Sequential treatment including nocodazole washout and removal of cell surface virion revealed that HCMV entry into the cells occurred normally in the absence of the MT network. This finding was also supported by data obtained by monitoring pUL83 signals with an immunofluorescent assay (IFA). Furthermore, we demonstrated a close association of incoming HCMV capsids with MTs by IFA and ultrastructural analyses. In the absence of the MT network, the capsids which had entered the cytoplasm did not move to close proximity of the nucleus. These data suggest that HCMV capsids associate with the MT network to facilitate their own movement to the nucleus before the onset of immediate-early (IE) gene expression and that this association is required to start efficient IE gene expression.

 

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.

Thirty-three patients with lung cancer receiving 80 mg m(-2) cisplatin were treated with high-dose dexamethasone (32 mg m(-2) on days 1-3, 16 mg m(-2) on day 4 and 8 mg m(-2) on day 5) combined with granisetron on day 1 and metoclopramide on days 2-5. Twenty-eight (85%) patients had no nausea or vomiting on day 1, and 16 (48%) achieved total control on days 1-5 with acceptable toxicity. High-dose dexamethasone for cisplatin-induced delayed emesis should be further evaluated in a phase III trial.

Cultivation of Candida albicans NIH B-792 (serotype B) at high temperature (37 degrees C) for 48 h in yeast extract-containing Sabouraud liquid medium (YSLM) provided the following findings in comparison with the findings obtained after incubation at 27 degrees C. Growth of the blastoconidia of this strain was decreased, with a dry weight of 9%, and the cells were deficient in cytokinesis. The cells did not undergo agglutination with serum factor 5 from a commercially available serum factor kit (Candida Check). Mannan (B-37-M) obtained from the cells cultured at 37 degrees C had partially lost its reactivity against serum factor 4 and lost most of its reactivity against serum factor 5 in an enzyme-linked immunosorbent assay (ELISA) in contrast to that (B-27-M) at 27 degrees C. Both cells and mannan prepared by cultivation first at 37 degrees C and then at 27 degrees C entirely recovered their reactivities with serum factors 4 and 5. 1H-nuclear magnetic resonance analysis also revealed that B-37-M had lost a beta-1,2-linked mannopyranose unit and retained a phosphate group. Similar changes were observed in the three other serotype B strains used in the study. The beta-1,2-linked mannooligosaccharides longer than mannotetraose were not included among the products released from B-37-M by mild acid treatment. The results of the inhibition ELISA with a series of beta-1,2-linked mannooligosaccharides from biose to octaose (M2 to M8, respectively) showed that the reactivity against serum factor 4 was inhibited most strongly by the oligosaccharides M4 to M8 and that the reactivity against serum factor 5 was inhibited completely by relatively longer oligosaccharides, M5 to M8, indicating their participation as the antigenic factor 5 epitopes.

AIMS/BACKGROUND--Recently HTLV-I has been shown to cause a kind of endogenous uveitis in south west Japan, where HTLV-I infection is highly endemic. To investigate further the association of HTLV-I infection with the incidence of this uveitis, HTLV-I seroprevalence in central Japan, where HTLV-I infection is not endemic, was studied. METHODS--HTLV-I seroprevalence was investigated in 1579 patients with various ocular diseases and 1251 normal volunteers as a younger control group. Then HTLV-I seroprevalence was compared in each group. RESULTS--Of 1579 patients with various ocular diseases, 38 (2.41%) were seropositive. There was a statistically significant difference in HTLV-I seroprevalence between the undefined uveitis group and non-uveitic ocular diseases group (p < 0.05, Yates's correction). However, the seroprevalence in younger patients with undefined uveitis did not differ significantly from that in other groups. As regards the incidence of this type of uveitis, six of 12 (50%) seropositive patients, who were born in south west Japan and had lived in this area for 35 years, developed this undefined uveitis whereas only two of 26 (7.69%) seropositive patients in the other areas in Japan developed this uveitis. The difference was statistically significant (p < 0.05, Fisher's exact probability test). CONCLUSION--These results suggest that the incidence of this type of endogenous uveitis could be greatly influenced by environmental or hereditary factors including HLA.

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The expression of the chicken delta 1-crystallin gene is primarily regulated by the action of a lens-specific enhancer 1 kilobase long and located in the third intron of the gene (S. Hayashi, K. Goto, T. S. Okada, and H. Kondoh, Genes Dev. 1:818-828, 1987). The 120-base-long core segment is required for the activity of the delta 1-crystallin enhancer but by itself shows no activity. We analyzed the action of the core and adjoining segments of the delta 1-crystallin enhancer by two different approaches: (i) multiplication of the segments to express any cryptic effect and (ii) competition among enhancers for nuclear factors involved in enhancer action. We found that (i) the core defines a strictly lens-specific element, (ii) an adjoining segment defines an element with a broad specificity with regard to cell type, (iii) these elements cooperate in cis within the delta 1-crystallin enhancer, (iv) the multimers of these elements complete with each other and with delta 1-crystallin and simian virus 40 enhancers in trans apparently without sequence specificity but in a fashion reflecting the strength of the enhancers, and (v) the enhancers in trans do not affect the expression of enhancer-free genes, thereby ruling out the possibility of competition for general transcription factors. The last two observations raise the possibility that the enhancer segments interacting with different sequence-specific factors also interact with one other component involved in enhancer action.

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The mechanisms of endothelin-1 (ET) actions were investigated in cultured rat aortic vascular smooth muscle A-10 cells. The A-10 cells have a single class of high affinity binding sites for ET with an apparent Mr of 65,000-75,000 on SDS-PAGE. Stimulation of cells with ET induces mobilization of Ca2+ from both intra- and extracellular pools to produce a biphasic increase in cytoplasmic free Ca2+ concentration. ET increases cellular levels of inositol trisphosphate and 1,2-diacylglycerol, indicating activation of phospholipase C by ET. ET stimulates production of inositol phosphates in membranes prepared from A-10 cells in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), but not in its absence. Further, specific binding of 125I-labeled ET to A-10 cell membranes is shown to be inhibited by GTP gamma S in a dose-dependent manner. Treatment of A-10 cells with pertussis toxin induces ADP-ribosylation of a 41,000-D membrane protein but fails to block the ET-induced increases in inositol phosphate production and Ca2+ mobilization. These results indicate that the receptor for ET is coupled to phospholipase C via a guanine nucleotide-binding regulatory protein which is distinct from the pertussis toxin substrate in A-10 cells.

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The correlation between the appearance of the characteristic electroencephalographic abnormality, cardinal clinical manifestations, and neuropathological features was studied in four cases of Creutzfeldt-Jakob syndrome consisting of subacute spongiform encephalopathy and classical Creutzfeldt-Jakob disease. Consideration is given to the differences in the electroencephalographic findings between these two subgroups of the syndrome and on the underlying pathological mechanism.

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The lens-specific reglatory element of the delta 1-crystallin enhancer lies within the core segment (Goto et al., (1990) Mol. Cell. Biol. 10, 935-964). The element was allocated within the 55 bp long HN fragment of the core. Block-wise base substitutions were introduced to the 55 bp and their effect on the enhancer activity of the multimers in lens cells was examined. By base sequence alteration of either of the contiguous blocks 5 and 6, with their original sequence of TTGCT and CACCT, respectively, enhancer activity was totally lost. A lens nuclear factor delta EF1 was found which bound specifically to the base sequences defined by the blocks. DNA binding activity very similar to delta EF1 was also found in extracts of tissues other than lens, suggesting that delta EF1 participates in lens-specific regulation through tissue-dependent modification or interaction with other factors.

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