Diffusion Tensor Imaging MRI With Spin-Echo Sequence and Long-Duration Measurement for Evaluation of Renal Fibrosis in a Rat Fibrosis Model.

BACKGROUND: Renal fibrosis (RF) is a well-known marker for chronic kidney disease (CKD) progression, including chronic renal injury after renal transplantation. However, invasive biopsy is an available examination for evaluation of RF. Diffusion MRI was once recognized as a promising option for RF. However, it is now controversial for RF evaluation in a unilateral ureteral obstruction (UUO) model. METHODS: To seek an optimal imaging method applicable for RF in UUO model kidneys, we attempted a series of MRI methods, including proton density-weighted imaging, T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging (DTI). RESULTS: We identified DTI MRI by spin-echo sequence plus a special kidney attachment as the best option for evaluation of renal UUO fibrosis, compared with normal kidney on the opposite side. To confirm these results, we applied this technique to a rat UUO therapeutic model with the anti-fibrotic reagent Fasudil. Fractional anisotropy values calculated from DTI MRI showed statistically significant linear correlation with the RF area measured by use of Sirius red or Masson trichrome staining of the positive area [cortex (r = 0.6397, P = .0283) and outer stripe of the outer medulla (r = 0.7810, P = .0039)]. CONCLUSIONS: By use of the DTI MRI with spin-echo sequence, it may be possible to accurately evaluate RF in CKD.

Quantification of retinal pigment epithelial phenotypic variation using laser scanning cytometry.

PURPOSE: Quantifying phenotypic variation at the level of protein expression (variegation) within populations of retinal pigment epithelium (RPE) cells may be important in the study of pathologies associated with this variation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicate this experimental goal. We have applied the technique of laser scanning cytometry (LSC) to paraffin sections of mouse and human eyes to evaluate the utility of LSC for these measurements. METHODS: Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Postmortem human eyes were fixed and dissected to obtain a 9-mm punch, which was then embedded in paraffin. A laser scanning cytometer equipped with violet, argon, and helium-neon lasers and the detectors for blue, green, and long red were used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using the WinCyte software. Individual nuclei were identified by the fluorescence of the 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Next, RPE cells were uniquely identified in the green channel using an anti-retinal pigment epithelium-specific protein 65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor 488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) was quantified in the long-red channel using an anti-MnSOD antibody and an Alexa Fluor 647-labeled secondary antibody. MnSOD(+) and RPE65(+) cells exhibited peaks in the plot of fluorescence intensity versus cell number, which could be characterized by the mean fluorescence intensity (MFI), the coefficient of variation (CV), and the percentage of total RPE cells that were also labeled for MnSOD. RESULTS: RPE cells can be uniquely identified in human and mouse paraffin sections by immunolabeling with anti-RPE65 antibody. A second antigen, such as MnSOD, can then be probed only within this set of RPE. Results are plotted primarily with the population frequency diagram, which can be subdivided into multiple regions. The data collected for each region include the MFI, the CV, and the number of cells that are immunolabeled in that region. Background interference from pigment or autofluorescent material can be successfully overcome by elevating the concentrations of fluorescent secondary antibodies. In the human and mouse eyes, age-related changes in MFI, CV, and percent RPE cells immunolabeled for MnSOD were observed. CONCLUSIONS: The extent of the variability of gene expression in RPE cells at the protein level can be quantified by LSC. Relative changes in the MFI, the CV, and/or percentage of RPE cells double labeled for a second antigen quantify the changes observed. The analysis of these data also suggest whether the effects observed are related to local changes in transcription (alterations of CV) or major changes of protein expression (MFI), which are likely to be due to changes in the chromatin structure. The changes of these variables with age suggest that the observed age-related variegation is primarily due to changes in the chromatin structure in individual cells.

Visualization of cervical nerve roots and their distal nerve fibers by diffusion-weighted scanning using parallel imaging.

PURPOSE: To report a technique developed for visualizing cervical nerve roots and distal nerve fibers using diffusion-weighted magnetic resonance imaging employing parallel imaging. MATERIAL AND METHODS: We performed maximum intensity projection for a stack of isotropic axial diffusion-weighted images obtained with parallel imaging applying a motion-probing gradient in six directions with a b-value of 500 s/mm2 in a preliminary series of 13 subjects. RESULTS: This method worked well for visualizing the spinal cord and most of the nerve roots, the dorsal root ganglia, and proximal peripheral nerves. CONCLUSION: Although the technique remains limited in depicting the brachial plexus and distal nerves, the ability to visualize the proximal peripheral nervous system at the cervical level is promising.

Differentiation between solitary brain metastasis and high-grade glioma by diffusion tensor imaging.

We tested our hypothesis that fractional anisotropy (FA) maps of diffusion tensor imaging could be used to differentiate between a solitary brain metastasis and a high-grade glioma. In seven patients with a solitary metastasis and seven patients with a high-grade glioma, FA values of enhancing and non-enhancing parts of the tumour were compared. Additionally, we visually assessed FA maps. No significant difference in the FA values of either the enhancing or non-enhancing part was found between the two groups. In the visual assessment, displacement of subcortical white-matter fibres was found in five of the seven metastasis patients, but in only one glioma patient. Additionally, discrimination between tumour and oedema was possible in three of the seven metastasis patients, but not in any glioma patient. Although FA values are not helpful in differentiating between the two groups, visual differences in FA values can allow the differentiation. Displacement of white-matter fibres is another finding suggestive of metastasis.

Early treatment for ruptured cerebral aneurysms. Location of aneurysms and choice of treatments.

SUMMARY: The purpose of this study was to evaluate the effect of endovascular treatment with Guglielmi detachable coils (GDC) on the outcomes of subarachnoid haemorrhage (SAH) patients of poor grades and high ages for each location of aneurysms. Between 1990 and 2003, 529 SAH cases underwent angiograghy as candidates of early aggressive treatment in our hospital. For the 299 cases in 1990-96 (Group 1), treatment options were early and intensively delayed craniotomy surgery and conservative management, while for the 230 cases in 1997-2003 (Group 2), GDG embolization at acute stage was added to these three treatment options. We compared clinical courses and outcomes of the poor grade (Hunt & Kosnik Grade 4-5) patients and high age (>=70 years old) patients between two groups for each location of aneurysms. Introduction of GDC embolization expanded the indication for early treatment in the poor grade patients with anterior communicating artery aneurysm (A-Comm An), the high age patients with internal carotid artery aneurysm (IC An) and all patients with Basilar bifurcation aneurysm (BA-Top An), and has contributed to improvement of their outcomes. To the poor grade patients with middle cerebral artery aneurysm (MCA An), GDC embolization was hardly indicated, because haematoma evacuation concomitantly performed with aneurysm occlusion would be necessary for those patients. In conclusion, results of treatment with GDC embolization at an acute stage are desirable for poor grade patients with A-Comm An, aged patients with IC An and all patients with BA-Top An. The indication of GDC embolization for the patients with MCA An is limited.

Diffusion-weighted MRI of subdural and epidural empyemas.

We reviewed diffusion-weighted images (DWI) from eight patients with subdural and four with epidural empyemas to assess the possibility of differentiating between these lesions by DWI. The signal intensities of the empyemas on DWI, and maps of the apparent diffusion coefficient (ADC) were analysed in seven patients. In seven of the eight patients with subdural empyema, the lesions appeared as areas of high signal. The ADC maps confirmed that these areas were the result of restricted diffusion. (In the remaining patient, the lesion showed a mixture of high and low signal.) The epidural empyemas contained areas of low signal in all four patients; part of the empyema was isointense or gave high signal in two. DWI may be an adjunct to conventional sequences for differentiating between subdural and epidural empyemas.

Diffusion-weighted MRI of the cervical spinal cord using a single-shot fast spin-echo technique: findings in normal subjects and in myelomalacia.

We have implemented a new diffusion-weighted MRI (DWI) sequence based on the single-shot fast spin-echo technique. We hypothesised that this would add information to conventional MRI for diagnosis of lesions of the cervical spinal cord. DWI was performed using a technique in which echo collection after the application of motion-probing gradients was done in the same manner as in the single-shot fast spin-echo technique. We first imaged six healthy volunteers to demonstrate the cervical spinal cord using the sequence. Then we applied the sequence to 12 patients with cervical myelomalacia due to chronic cord compression. The spinal cord was well seen in all subjects without the distortion associated with echo-planar DWI. In the patients, lesions appeared as areas of low- or isointense signal on DWI. Calculated apparent diffusion coefficients of the lesions (3.30+/-0.38x10(-3) mm(2)/s) were significantly higher than those of normal volunteers (2.26+/-0.08x10(-3) mm(2)/s). Increased diffusion in areas of cervical myelomalacia, suggesting irreversible damage, can be detected using this technique.

Diffusion-weighted MR imaging of Carmofur-induced leukoencephalopathy.

Carmofur (1-hexylcarbamyl-5-fluorouracil), a derivative of 5-fluorouracil (5-FU), has been widely used in Japan as a postoperative adjuvant chemotherapy agent for colorectal and breast cancer. Periventricular hyperintensity on T2-weighted MR images in carmofur-induced leukoencephalopathy confront the physician with a broad range of differential diagnoses. We describe two cases of carmofur-induced leukoencephalopathy in which diffusion-weighted MR imaging revealed periventricular hyperintensity. We compared their findings with those of age-related periventricular hyperintensity in five patients and found discrepancies in signal intensity of periventricular areas. Our results suggest that diffusion-weighted MR imaging may be useful to differentiate carmofur-induced leukoencephalopathy from age-related periventricular hyperintensity.

Crystals of ternary protein-DNA complexes composed of DNA-binding domains of c-Myb or v-Myb, C/EBPalpha or C/EBPbeta and tom-1A promoter fragment.

c-Myb and the C/EBP family are transcriptional regulatory factors that act in concert to regulate the expression of myeloid-specific genes. v-Myb encoded by avian myeloblastosis virus (AMV) is a mutated form of c-Myb that contains point mutations which disrupt the cooperation with C/EBPs. To understand the mechanism of the transcriptional synergy between c-Myb and C/EBPs and the effect of the v-Myb mutations on that synergy, knowledge based on their three-dimensional structures is essential. Crystals of ternary complexes, in which various combinations of the DNA-binding domains of c-Myb or v-Myb and C/EBPalpha or C/EBPbeta are bound to a DNA fragment from tom-1A promoter, were obtained by the vapour-diffusion method. Complete diffraction data sets were obtained from each native crystal and two types of iodine-derivative crystals. A three-wavelength MAD data set was also obtained from a bromine-derivative crystal.

Identification of GIT1/Cat-1 as a substrate molecule of protein tyrosine phosphatase zeta /beta by the yeast substrate-trapping system.

We used a genetic method, the yeast substrate-trapping system, to identify substrates for protein tyrosine phosphatases zeta (PTPzeta/RPTPbeta). This method is based on the yeast two-hybrid system, with two essential modifications: conditional expression of protein tyrosine kinase v-src (active src) to tyrosine-phosphorylate the prey proteins and screening by using a substrate-trap mutant of PTPzeta (PTPzeta-D1902A) as bait. By using this system, several substrate candidates for PTPzeta were isolated. Among them, GIT1/Cat-1 (G protein-coupled receptor kinase-interactor 1/Cool-associated, tyrosine-phosphorylated 1) was examined further. GIT1/Cat-1 bound to PTPzeta-D1902A dependent on the substrate tyrosine phosphorylation. Tyrosine-phosphorylated GIT1/Cat-1 was dephosphorylated by PTPzeta in vitro. Immunoprecipitation experiments indicated that PTPzeta-D1902A and GIT1/Cat-1 form a stable complex also in mammalian cells. Immunohistochemical analyses revealed that PTPzeta and GIT1/Cat-1 were colocalized in the processes of pyramidal cells in the hippocampus and neocortex in rat brain. Subcellular colocalization was further verified in the growth cones of mossy fibers from pontine explants and in the ruffling membranes and processes of B103 neuroblastoma cells. Moreover, pleiotrophin, a ligand for PTPzeta, increased tyrosine phosphorylation of GIT1/Cat-1 in B103 cells. All these results indicate that GIT1/Cat-1 is a substrate molecule of PTPzeta.

Early and delayed neuroprotective effects of FK506 on experimental focal ischemia quantitatively assessed by diffusion-weighted MRI.

The immunosuppressive drug FK506 (tacrolimus) has been reported to be a powerful neuroprotective agent in the focal ischemia of animals. However, no report has been published concerning neuroprotective effect of this compound on the morphology in superacute stage. The separate analysis between early and delayed effects of FK506 on the morphology may be helpful in the study of the compound's mechanism of action which is still unknown. The goal of this study was to determine early and delayed effects of pharmacological treatment with FK506 in permanent MCA occlusion using magnetic resonance imaging (MRI). Nineteen rats were subjected to permanent MCA occlusion, and given either intravenous injection of placebo or 1 mg/kg FK506 immediately after occlusion. DWI and T(2)-weighted MRI were performed 3 and 24 h after MCA occlusion, and postmortem histological analysis was also performed. FK506 drastically reduced the ischemic damage in 3-h apparent diffusion coefficient (ADC) map. This is the first report to demonstrate the neuroprotective effects of FK506 on focal cerebral ischemia in superacute stage. In addition, postmortem ischemic damage tended to be smaller than ischemic area indicated by 3-h ADC map in the FK506 group, whereas there was an excellent equality between them in the placebo group, suggesting the possible effect of FK506 on the later ischemic period. Our findings provide direct evidence for the neuroprotective effect of FK506 on ischemic cell damage in both early stage and possibly later stage.

Crystallization and preliminary X-ray analysis of the C/EBPbeta C-terminal region in complex with DNA.

The C-terminal fragment (residues 259-345) of human C/EBPbeta, a basic region leucine zipper transcriptional regulatory factor which includes the minimal DNA-binding domain, was crystallized in complex with a 16 bp DNA fragment from the tom-1 promoter. The crystals were in the form of a parallelepiped belonging to space group C222(1), had unit-cell parameters a = 100.7 (2), b = 113.5 (1), c = 74.4 (1) A and diffracted to a resolution of 2.1 A. Moreover, truncation of nine residues from the C-terminus not conserved among C/EBP family members yielded isomorphous crystals that diffracted to a resolution of 1.8 A or better. Truncation of 14 residues from the N-terminus of the C-terminal fragment produced well shaped crystals in the form of hexagonal bipyramids, however; unfortunately, they were unstable and diffracted poorly.

Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its allosteric control by CBFbeta.

The core binding factor (CBF) heterodimeric transcription factors comprised of AML/CBFA/PEBP2alpha/Runx and CBFbeta/PEBP2beta subunits are essential for differentiation of hematopoietic and bone cells, and their mutation is intimately related to the development of acute leukemias and cleidocranial dysplasia. Here, we present the crystal structures of the AML1/Runx-1/CBFalpha(Runt domain)-CBFbeta(core domain)-C/EBPbeta(bZip)-DNA, AML1/Runx-1/CBFalpha(Runt domain)-C/EBPbeta(bZip)-DNA, and AML1/Runx-1/CBFalpha(Runt domain)-DNA complexes. The hydrogen bonding network formed among CBFalpha(Runt domain) and CBFbeta, and CBFalpha(Runt domain) and DNA revealed the allosteric regulation mechanism of CBFalpha(Runt domain)-DNA binding by CBFbeta. The point mutations of CBFalpha related to the aforementioned diseases were also mapped and their effect on DNA binding is discussed.

Nav2/NaG channel is involved in control of salt-intake behavior in the CNS.

Na(v)2/NaG is a putative sodium channel, whose physiological role has long been an enigma. We generated Na(v)2 gene-deficient mice by inserting the lacZ gene. Analysis of the targeted mice allowed us to identify Na(v)2-producing cells by examining the lacZ expression. Besides in the lung, heart, dorsal root ganglia, and Schwann cells in the peripheral nervous system, Na(v)2 was expressed in neurons and ependymal cells in restricted areas of the CNS, particularly in the circumventricular organs, which are involved in body-fluid homeostasis. Under water-depleted conditions, c-fos expression was markedly elevated in neurons in the subfornical organ and organum vasculosum laminae terminalis compared with wild-type animals, suggesting a hyperactive state in the Na(v)2-null mice. Moreover, the null mutants showed abnormal intakes of hypertonic saline under both water- and salt-depleted conditions. These findings suggest that the Na(v)2 channel plays an important role in the central sensing of body-fluid sodium level and regulation of salt intake behavior.

Choroidal detachment after vitreous surgery.

BACKGROUND AND OBJECTIVE: To study the frequency and clinical features of choroidal detachment (CD) after vitreous surgery, because there have been no reports on this problem. PATIENTS AND METHODS: We studied the clinical features of 14 patients (15 eyes) with CD from a total of 380 patients treated with vitrectomy at the Nagasaki University Hospital from January 1994 to August 1997. RESULTS: The incidence of CD after vitreous surgery was 3.9% (15/380). The reasons for vitrectomy were 6 retinal detachments, 4 proliferative diabetic retinopathies, and 5 others. During vitrectomy, 4 eyes were treated with scleral buckling, 11 with endolaser photocoagulation, and 3 with cryoretinopexy. Retinal detachment as a postoperative complication was seen in 8 patients, and in 5 of them the retina remained detached after the final treatment. CONCLUSIONS: CD may be caused by scleral buckling, panphotocoagulation, or stress on the ciliary body. Some patients with CD have a poor outcome.

1H-NMR determination of the solution structure and absolute configuration of FR134043, a novel inhibitor of human leukocyte elastase.

FR134043 is a semisynthetic disulfonated derivative of the natural product FR901277, is isolated from the culture filtrate of Streptomyces resistomicificus and has potent inhibitory activity against human leukocyte elastase. Although the chemical structure of FR134043 was determined to be a unique bicyclic peptide-like compound consisting of seven amino acids by using several spectroscopic analytical methods, the chiralities of three centers were unknown. A simple simulated annealing protocol to determine the structure was applied to the eight possible stereoisomers, and the one that best satisfied the NOE distance constraints was determined to be the true stereoconfiguration of FR134043. The solution structure showed that all Calpha atoms existed in the L configuration and six of the seven side chains were located towards the outside of the bicyclic framework, even though most of them are highly hydrophobic moieties. The simulated annealing calculation described here is a frequently used method for the determination of the solution structure of peptides or small proteins. We show here that it is also applicable to the determination of the absolute configuration of macrocyclic compounds produced from natural sources.

Diagnosis of the nutcracker syndrome with color Doppler sonography: correlation with flow patterns on retrograde left renal venography.

OBJECTIVE: Our objective was to confirm flow patterns of the left renal vein and assess the usefulness of color Doppler sonography in the diagnosis of the renal vein entrapment, or nutcracker, syndrome. SUBJECTS AND METHODS: Forty-four patients with hematuria of unknown origin underwent color Doppler sonography, renal arteriography, retrograde left renal venography, and renocaval pressure measurement. The flow patterns were classified according to the presence or absence of distended left renal veins on sonograms and collateral veins on retrograde venograms. The sensitivity and specificity of color Doppler sonography for revealing the nutcracker syndrome were assessed by analyzing pressure gradients in combination with the venograms, which were used as the gold standard. RESULTS: Twenty-one nondistended left renal veins were seen, including 19 (43%) without collateral veins or hypertension and two (5%) with collateral veins but without hypertension (long-term nutcracker syndrome). The 23 distended left renal veins included seven without collateral veins or hypertension and one with hypertension but without collateral veins (early nutcracker syndrome). The remaining 15 distended left renal veins with collateral veins included 11 (25%) with borderline hypertension (partially compensatory status of the syndrome) and four (9%) with hypertension (noncompensatory status of the syndrome). The sensitivity and specificity of color Doppler sonography for revealing the nutcracker syndrome were 78% and 100%, respectively, when color flow in collateral veins was included in the diagnostic criteria. CONCLUSION: The nutcracker syndrome can exist in either nondistended or distended left renal veins. However, normal flow also can exist in distended left renal veins. The ability of color Doppler sonography to reveal color flow in collateral veins is useful for the noninvasive diagnosis of the nutcracker syndrome.

Altered antigenicity of M-177, a 177-kDa allergen from the house dust mite Dermatophagoides farinae, in stored extract.

BACKGROUND: A high molecular weight allergen, M-177 (177 kDa) was isolated from Dermatophagoides farinae using a specific antibody raised to an allergenic clone Mag 3, which was obtained by immunoscreening a mite cDNA library. The potent IgE reactivity of M-177 is comparable with that of Der f 2. OBJECTIVE: The aim of this study was to analyse the molecular characteristics and the allergenic activity of M-177 in stored mite extracts. METHODS: Antigens were analysed by immunoblotting and enzyme-linked immunosorbent assay (ELISA; inhibition). Allergenic activity was estimated from IgE reactivity and the results of a histamine release assay. RESULTS: The intact M-177 molecule was present in high concentrations in fresh extract obtained from purified mite bodies, but was only detected in small amounts in stored extracts. Instead of the intact molecule, anti-Mag 3 antibody detected various cross-reactive antigens in the stored preparations. Studies of a stored liquid extract showed that these cross-reactive antigens were produced by the degradation of M-177, and that this change was suppressed by the addition of protease inhibitors. Interestingly, the allergenic activity of the fragmented M-177 (sM-177) isolated from the stored extracts was greater than that of the intact antigen. Specific IgE reacted with sM-177 in 84.2% of 38 sera samples from patients allergic to mites, while 65.8% were positive for M-177-specific IgE. Similarly, the histamine release test showed that sM-177 had greater allergenic activity in vitro. ELISA inhibition indicated that the increased allergenic activity resulted from alteration of the antigenicity with the degradation of M-177. CONCLUSIONS: M-177 is a protease-sensitive allergen. The breakdown products of M-177 provoked higher allergenic activity than the intact allergen.

Cloning and characterization of a new allergen, Mag 3, from the house dust mite, Dermatophagoides farinae: cross-reactivity with high-molecular-weight allergen.

A new immunoreactive clone whose sequence is not homologous to that of any of the previously identified mite allergens was isolated by successive immunoscreening of a Dermatophagoides farinae cDNA library with rabbit antisera to an extract of the house dust mite and IgE in pooled sera from patients allergic to mites. Rabbit antibodies specific for the recombinant protein recognized a 177 kD protein in a mite body extract. This immunoreactive protein was located in the circumferential tissues of esophagus, gut and the other internal organs in mites. The reaction of human IgE to the purified natural antigen was inhibited competitively to 30% by the recombinant antigen. In terms of the frequency and the intensity of response to specific IgE in sera from asthmatic patients, the natural protein was similar to Der f2, while the recombinant protein was slightly less allergenic by these criteria. We conclude that the natural protein from the house dust mite, D. farinae, is an important allergen.