AKAP350 at the Golgi apparatus. II. Association of AKAP350 with a novel chloride intracellular channel (CLIC) family member

AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. We performed a yeast two-hybrid screen of a rabbit parietal cell library with a 3.2-kb segment of AKAP350 (nucleotides 3611-6813). This screen yielded a full-length clone of rabbit chloride intracellular channel 1 (CLIC1). CLIC1 belongs to a family of proteins, all of which contain a high degree of homology in their carboxyl termini. All CLIC family members were able to bind a 133-amino acid domain within AKAP350 through the last 120 amino acids in the conserved CLIC carboxyl termini. Antibodies developed against a bovine CLIC, p64, immunoprecipitated AKAP350 from HCA-7 colonic adenocarcinoma cell extracts. Antibodies against CLIC proteins recognized at least five CLIC species including a novel 46-kDa CLIC protein. We isolated the human homologue of bovine p64, CLIC5B, from HCA-7 cell cDNA. A splice variant of CLIC5, the predicted molecular mass of CLIC5B corresponds to the molecular mass of the 46-kDa CLIC immunoreactive protein in HCA-7 cells. Antibodies against CLIC5B colocalized with AKAP350 at the Golgi apparatus with minor staining of the centrosomes. AKAP350 and CLIC5B association with Golgi elements was lost following brefeldin A treatment. Furthermore, GFP-CLIC5B-(178-410) targeted to the Golgi apparatus in HCA-7 cells. The results suggest that AKAP350 associates with CLIC proteins and specifically that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells.     

Scarce adenylation in bacteriophage T4 mRNAs

The degradation of mRNA is crucial for the rapid shift of bacteriophage T4 gene expression from early to late. The present study was conducted to investigate whether T4 mRNA is polyadenylated or not, because polyadenylation is known to facilitate the degradation of mRNA in Escherichia coli cells. Total RNA extracted from T4-infected cells was subjected to self-circularization or intermolecular ligation by T4 RNA ligase, and a region containing the 3'-5' junction was amplified by RT-PCR. Cloning and sequencing as well as the length distribution of amplified DNA fragments revealed no adenines at the 3'-ends of uvsY and soc RNAs. The present result suggests that T4 mRNA is not significantly adenylated.     

Intratumoral injection of dendritic and irradiated glioma cells induces anti-tumor effects in a mouse brain tumor model

Malignant astrocytoma is the most common primary brain tumor in adults. The median survival of patients with malignant astrocytomas (high-grade astrocytomas) is about 1-2 years, despite aggressive treatment that includes surgical resection, radiotherapy and cytotoxic chemotherapy. Therefore, novel therapeutic approaches are needed to prolong survival. We investigated antitumor immunity conferred by the intratumoral injection of dendritic (DC) and irradiated glioma cells (IR-GC) in a mouse brain tumor model. Intratumorally injected DC migrated to the lymph nodes and elicited systemic immunity against autologous glioma cells. In a treatment model, intratumoral injection of DC and IR-GC prolonged the survival of brain tumor-bearing mice. Efficacy was reduced when studies were performed in mice depleted of CD8(+) T cells. Administration of DC or IR-GC alone had no effect on survival of brain tumor-bearing mice. CTL activity against glioma cells from immunized mice was also stimulated by coadministration of DC and IR-GC compared with the controls. These results support the therapeutic efficacy of intratumoral injection of DC and IR-GC.     

The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.     

One-dimensional viscoelastic behavior of fibroblast populated collagen matrices

Bio-artificial tissues are being developed as replacements for damaged biologic tissues. Their mechanical properties are critical for load bearing applications. Current testing protocols for bio-artificial tissues vary widely and often do not consider viscoelasticity. Uniaxial stretch tests were performed on fibroblast populated collagen matrices (FPCMs) to determine the influence of specific test protocols on the mechanical behavior. The peak force, hysteresis and shape of the force-stretch curve are affected by the stretch rate, rest period, stretch amplitude and the number and magnitude of preconditioning cycles.     

Assessing chimpanzee personality and subjective well‐being in Japan

We tested whether the cultural background of raters influenced ratings of chimpanzee personality. Our study involved comparing personality and subjective well‐being ratings of 146 chimpanzees in Japan that were housed in zoos, research institutes, and a retirement sanctuary to ratings of chimpanzees in US and Australian zoos. Personality ratings were made on a translated and expanded version of a questionnaire used to rate chimpanzees in the US and Australia. Subjective well‐being ratings were made on a translated version of a questionnaire used to rate chimpanzees in the US and Australia. The mean interrater reliabilities of the 43 original adjectives did not markedly differ between the present sample and the original sample of 100 zoo chimpanzees in the US. Interrater reliabilities of these samples were highly correlated, suggesting that their rank order was preserved. Comparison of the factor structures for the Japanese sample and for the original sample of chimpanzees in US zoos indicated that the overall structure was replicated and that the Dominance, Extraversion, Conscientiousness, and Agreeableness domains clearly generalized. Consistent with earlier studies, older chimpanzees had higher Dominance and lower Extraversion and Openness scores. Correlations between the six domain scores and subjective well‐being were comparable to those for chimpanzees housed in the US and Australia. These findings suggest that chimpanzee personality ratings are not affected by the culture of the raters. Am. J. Primatol. 71:283–292, 2009. © 2009 Wiley‐Liss, Inc.     

Quantitative Analysis of Torso FDG-PET Scans by Using Anatomical Standardization of Normal Cases from Thorough Physical Examinations

Understanding of standardized uptake value (SUV) of 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) depends on the background accumulations of glucose because the SUV often varies the status of patients. The purpose of this study was to develop a new method for quantitative analysis of SUV of FDG-PET scan images. The method included an anatomical standardization and a statistical comparison with normal cases by using Z-score that are often used in SPM or 3D-SSP approach for brain function analysis. Our scheme consisted of two approaches, which included the construction of a normal model and the determination of the SUV scores as Z-score index for measuring the abnormality of an FDG-PET scan image. To construct the normal torso model, all of the normal images were registered into one shape, which indicated the normal range of SUV at all voxels. The image deformation process consisted of a whole body rigid registration of shoulder to bladder region and liver registration and a non-linear registration of body surface by using the thin-plate spline technique. In order to validate usefulness of our method, we segment suspicious regions on FDG-PET images manually, and obtained the Z-scores of the regions based on the corresponding voxels that stores the mean and the standard deviations from the normal model. We collected 243 (143 males and 100 females) normal cases to construct the normal model. We also extracted 432 abnormal spots from 63 abnormal cases (73 cancer lesions) to validate the Z-scores. The Z-scores of 417 out of 432 abnormal spots were higher than 2.0, which statistically indicated the severity of the spots. In conclusions, the Z-scores obtained by our computerized scheme with anatomical standardization of torso region would be useful for visualization and detection of subtle lesions on FDG-PET scan images even when the SUV may not clearly show an abnormality.     

Blood diagnostic biomarkers for major depressive disorder using multiplex DNA methylation profiles: discovery and validation

Aberrant DNA methylation in the blood of patients with major depressive disorder (MDD) has been reported in several previous studies. However, no comprehensive studies using medication-free subjects with MDD have been conducted. Furthermore, the majority of these previous studies has been limited to the analysis of the CpG sites in CpG islands (CGIs) in the gene promoter regions. The main aim of the present study is to identify DNA methylation markers that distinguish patients with MDD from non-psychiatric controls. Genome-wide DNA methylation profiling of peripheral leukocytes was conducted in two set of samples, a discovery set (20 medication-free patients with MDD and 19 controls) and a replication set (12 medication-free patients with MDD and 12 controls), using Infinium HumanMethylation450 BeadChips. Significant diagnostic differences in DNA methylation were observed at 363 CpG sites in the discovery set. All of these loci demonstrated lower DNA methylation in patients with MDD than in the controls, and most of them (85.7%) were located in the CGIs in the gene promoter regions. We were able to distinguish patients with MDD from the control subjects with high accuracy in the discriminant analysis using the top DNA methylation markers. We also validated these selected DNA methylation markers in the replication set. Our results indicate that multiplex DNA methylation markers may be useful for distinguishing patients with MDD from non-psychiatric controls.