Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein.

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C zeta (PKCzeta) as a bait, we have cloned a gene coding for a novel PKCzeta-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCzeta and weakly with that of PKCepsilon. In the COS-7 cells coexpressing FEZ1 and PKCzeta, FEZ1 was present mainly in the plasma membrane, associating with PKCzeta and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCzeta. When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCzeta activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCzeta stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCzeta.

Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways.

In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.

A Qualitative and Quantitative Correlation Study of Lumbar Intervertebral Disc Degeneration Using Glycosaminoglycan Chemical Exchange Saturation Transfer, Pfirrmann Grade, and T1-ρ.

BACKGROUND AND PURPOSE: Glycosaminoglycan chemical exchange saturation transfer (gagCEST) imaging allows the direct measurement and mapping of glycosaminoglycans. In this study, we aimed to evaluate the usefulness of gagCEST imaging in the quantitative assessment of intervertebral disc degeneration in a comparison with Pfirrmann grade and T1-ρ measurements. MATERIALS AND METHODS: Ninety-six lumbar intervertebral discs in 24 volunteers (36.0 ± 8.5 years of age, 21 men and 3 women) were examined with both gagCEST imaging and T1-ρ measurements. The gagCEST imaging was performed at 3T with a saturation pulse with 1.0-second duration and the B1 amplitude of 0.8 µT followed by imaging by a 2D fast spin-echo sequence. The Z-spectra were obtained at 25 frequency offsets from -3 to +3 ppm (step, 0.25 ppm). A point-by-point B0 correction was performed with a B0 map. The gagCEST signal and T1-ρ values were measured in the nucleus pulposus in each intervertebral disc. The Pfirrmann grades were assessed on T2-weighted images. RESULTS: The gagCEST signal at grade I (5.36% ± 2.79%) was significantly higher than those at Pfirrmann grade II (3.15% ± 1.40%, P = .0006), grade III (0.14% ± 1.03%, P < .0001), grade IV (-1.75% ± 2.82%, P < .0001), and grade V (-1.47% ± 0.36%, P < .0001). The gagCEST signal at grade II was significantly higher than those of grade III (P < .0001), grade IV (P < .0001), and grade V (P < .0001). The gagCEST signal was significantly correlated negatively with Pfirrmann grade (P < .0001) and positively correlated with T1-ρ (P < .0001). CONCLUSIONS: GagCEST imaging could be a reliable and quantitative technique for assessing intervertebral disc degeneration.

Kinase activities associated with mTOR.

Although mTOR is a member of the PI-kinase-related kinase family, mTOR possesses serine-threonine protein kinase activities, which phosphorylate itself and exogenous substrates. mTOR autophosphorylates in vitro and is phosphorylated in vivo on serine residues. Ser2481, which is located in a His-Ser-Phe motif near the conserved carboxyl-terminal mTOR tail, has been reported as an autophosphorylation site in vivo and in vitro. The significance of the autophosphorylation remains unclear. Another phosphorylation site on mTOR in vivo is Ser2448. This site appears not to be an autophosphorylation site but a site potentially phosphorylated by protein kinase B (PKB). mTOR immunopurified from culture cells or tissues phosphorylates in vitro p70 S6 kinase (p70) alpha and p70beta, mainly on Thr412 or Thr401, respectively, located in a Phe-Thr-Tyr motif. Another exogenous substrate phosphorylated by immunopurified mTOR in vitro is eIF4E-binding protein 1 (4E-BP1) at sites corresponding to those phosphorylated in vivo during insulin stimulation in a Ser/Thr-Pro motif. Recently, raptor, a 150-kDa TOR-binding protein that contains a carboxyl-terminal WD-repeat domain, was discovered as a scaffold for the mTOR-catalyzed phosphorylation of 4E-BP1 and for the mTOR-mediated phosphorylation and activation of p70alpha. Other potential substrates phosphorylated by mTOR are nPKCdelta, nPKCepsilon, STAT3, and p53. The requirement of raptor for binding to and phosphorylation by mTOR of these potential substrates would clarify their physiological importance in the mTOR signaling pathway.

[Asplenia syndrome with total anomalous pulmonary venous connection].

Three patients of asplenia syndrome with total anomalous pulmonary venous connection (TAPVC) were reported. Case 1 with exceeding pulmonary blood flow, underwent TAPVC repair and pulmonary artery banding as a first palliation before bi-directional Glenn shunt. Case 2 did not require any surgery to control the pulmonary blood flow before the simultaneous procedure of TAPVC repair and bi-directional Glenn shunt. Case 3 with decreased pulmonary blood flow underwent a complicated course with 3 times of Blalock-Taussig shunts and the repair of TAPVC to prepare for bi-directional Glenn shunt. Simultaneous repair of TAPVC with the procedure which aimed to control the pulmonary blood flow at the first palliation surgery will simplify the control of pulmonary blood flow and prepare good condition of the lung for the Fontan operation in the future.

[Delayed onset malignant hyperthermia after a closure of ventricular septal defect].

An 18 month-old girl was diagnosed as ventricular septal defect (VSD) with mild aortic valve prolapse. She underwent a closure of VSD. Intra-and early postoperative course was uneventful. However, 20 hours after surgery, sudden bradycardia led to cardiac arrest and strong muscle rigidity was seen. Hyperkalemia and metabolic acidosis rapidly progressed and resuscitation was failed. Extracorporeal life support and continuous hemodialysis were initiated, but the patient died with multiple organ failure on 5th postoperative day. Her clinical course supported the diagnosis of delayed onset malignant hyperthermia. Histopathological findings of muscle biopsy were consistent with rhabdomyolysis, and immunopathological stains demonstrated changes as in a Duchenne type muscular dystrophy carrier. Delayed onset malignant hyperthermia is an extremely rare complication of general anesthesia. We should be aware of this lethal condition, which occurs with a certain time lag after surgery, especially when the patient has possible background of myopathy.

[Cor triatriatum diagnosed in adulthood; report of a case].

Cor triatriatum is a rare congenital cardiac anomaly especially in adulthood. A 68-year-old female was diagnosed as a cor triatriatum classified to Lucas-Schmidt IA, severe degree of mitral regurgitation and atrial fibrillation. Resection of the abnormal diaphragm in the left atrium and miral valve replacement were performed. Although the reason of sudden death of this patient after discharge is unknown, surgical intervention for atrial fibrillation should have performed to prevent a thromboembolism in such cases.

Molecular cloning and characterization of RBCK2, a splicing variant of a RBCC family protein, RBCK1.

RBCK1 (RBCC protein interacting with PKC 1) has two coiled-coil regions, a RING finger, a B-box and a B-box-like motif. RBCK2, a cDNA fragment related to RBCK1 was obtained, that lacks the 161-bp sequence of RBCK1 and encodes 260 amino acid residues. The 240-amino acid sequence in the NH2-terminal of RBCK2 is identical with RBCK1 and contains two coiled-coil regions but no other structural motifs, whereas the 20-amino acid sequence in the COOH-terminal is distinct from RBCK1. The analysis of genomic DNA revealed that RBCK1 and RBCK2 are generated from a single gene by alternative splicing. The RBCK1 protein interacted with the RBCK1 and RBCK2 proteins, but the RBCK2 protein did not interact with itself, in vitro. The RBCK2 protein fused with the DNA-binding domain of yeast GAL4 (GAL4DBD) did not show a transcriptional activity, but the RBCK2 protein inhibited the transcriptional activity of the RBCK1 protein fused with GAL4DBD. These results suggest that RBCK2 may inhibit the transcriptional activity of RBCK1 probably through complex formation with RBCK1.

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases.

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.

[Leaflet extension technique in a congenital aortic valve regurgitation].

A 17-year-old boy who was diagnosed as congenital aortic valve regurgitation underwent the leaflet extension technique in 3 aortic leaflets using glutaraldehyde-preserved autologous pericardium. Intraoperative transesophageal echocardiography and postoperative cardiac catheter revealed grade I regurgitation and the patient has been doing well for more than 4 months after the surgery. The leaflet extension technique is considered to be an acceptable surgical treatment for aortic valve disease especially in young patients to whom valve replacement or Ross procedure might not be suitable. A careful long-term follow-up will be necessary to evaluate the durability of the leaflet extension technique with autologous pericardium.

[Management of mediastinitis after pediatric cardiac surgery with continuous closed irrigation method].

Two cases are presented as a successful management for mediastinitis with the continuous closed irrigation method after pediatric cardiac surgery. The continuous closed irrigation method has significant advantages over conventional open irrigation method or muscle flap in pediatric cases, because the system is simple to handle and easy to wash out any infectious tissue debris without additional invasive intervention. However, adequate duration of the irrigation and subsequent antibiotic regimen is still unclear. We conclude that the continuous closed irrigation method is an effective management which is applicable to most of mediastinitis cases after pediatric cardiac surgery.

TU-E-BRB-01: Similar-Case-Based Optimization of Beam Arrangements in Stereotactic Body Radiation Therapy.

PURPOSE: The quality of a treatment plan for stereotactic body radiotherapy (SBRT) depends on an experience of each treatment planner. Therefore, the treatment plans are subjectively determined by comparison of several treatment plans developed by time consuming iterative manners, while considering the benefit to a tumor and the risk to the surrounding normal tissues. The aim of our study was to develop an automated optimization method for beam arrangements based on similar cases in a database including plans designed by senior experienced treatment planners. METHODS: Our proposed method consists of three steps. First, similar cases were automatically selected based on image features from the treatment planning point of view. We defined four types of image features relevant to planning target volume (PTV) location, PTV shape, lung size, and spinal cord positional features. Second, the beam angles of the similar case were registered to the objective case with respect to lung regions using a linear registration technique. Third, the beam direction of the objective case was locally optimized based on the cost function considering radiation absorption in normal tissues and organs at risk. The proposed method was evaluated with 10 test cases and a treatment planning database including 81 cases by using eight planning evaluation indices such as D95, lung V20, and maximum spinal cord dose. RESULTS: The proposed method may provide usable beam directions, which have no statistically significant differences with the original beam directions (P > 0.05) in terms of the seven planning evaluation indices. Moreover, the mean value of D95 for 10 test cases was improved with a statistically significant difference by using the proposed method, compared with the original beam directions (P = 0.03). CONCLUSIONS: The proposed method could be used as a computer-assisted treatment planning tool for determination of beam directions in SBRT.

Changes in chemical and sensory properties of migaki-nishin (dried herring fillet) during drying.

Migaki-nishin is a Japanese term that refers to dried herring fillets. It is widely consumed in Japan due to its characteristic flavor enhancing properties. This study was conducted to investigate the changes in chemical and sensory properties of migaki-nishin during drying. Chemical analyses showed that extractive nitrogen and amount of peptides increased significantly (P < 0.05) up to the 8th day of drying and then slightly decreased by the 10th day. Glutamic acid, alanine, glycine, and histidine were the most abundant free amino acids and the largest increase was found in samples dried for 10 d. A decrease in Hunter's L* value (lightness) and increase in b* value (yellowness) as well as browning intensity suggested that nonenzymatic browning occurred in migaki-nishin during drying. Fluorescence spectrophotometric determination also revealed that Maillard reactions progressed throughout the drying period. In addition, available lysine content and free amino groups decreased significantly (P < 0.05) as drying progressed. Sensory evaluation showed that addition of water-soluble extracts to Japanese noodle soup (mentsuyu) linearly enhanced the flavor characteristics such as thickness, mouthfulness, and continuity with the increased length of drying time. These results suggest that during the drying period, proteolysis as well as Maillard reaction products increased markedly, which might contribute to the characteristic taste and flavor of migaki-nishin.

Prevalencia de las maloclusiones en el Departamento de Ortodoncia de la División de Estudios de Postgrado e Investigación de la Facultad de Odontología de la Universidad Nacional Autónoma de México / Prevalence of malocclusions at the Orthodontics Department of the Graduate School, National School of Dentistry, National University of Mexico (UNAM)

En la División de Estudios de Postgrado e Investigación de la Universidad Nacional Autónoma de México, en el Departamento de Ortodoncia, nosotros desarrollamos diferentes métodos analíticos y descriptivos, donde se realizó estudio estadístico de la clasificación esqueletal con una muestra de 428 pacientes que recibieron tratamiento de ortodoncia. Se seleccionaron personas entre 8 y 40 años de edad. Se capturaron datos de acuerdo a sexo, edad y maloclusión esqueletal para conocer el panorama epidemiológico. Después del análisis estadístico encontramos que el 53.3% de la muestra se encontraba, en clase I esqueletal, que el 64.7% era del sexo femenino y que el 52.08% se encontraba en el rango de edad de 13 a 19 años, además de otros datos.

At the Graduate and Research School of the National School of Dentistry, National University of Mexico (UNAM) we developed several analytic and descriptive methods. A statistical study of skeletal classification was undertaken with a sample of 428 patients subjected to orthodontic treatment. Age range of selected patients was 8-40 years. Data were collected according to gender, age and skeletal malocclusion in order to assess the epidemiological panorama. After statistical analysis, it was found among other data, that 53.3% of the sample was in skeletal class I, 64.7% were female and 52.08% was found to be in the 13 to 19 year age range.

Evidence for impaired gluconeogenesis in very long-chain acyl-CoA dehydrogenase-deficient mice.

Hypoketotic hypoglycaemia is a characteristic feature of fatty acid oxidation (FAO) defects. Although the underlying pathogenic mechanism is unknown, one hypothesis points to an impairment in gluconeogenesis. To study hepatic glucose production in FAO defects, we used the knockout mouse model of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency presenting with stress-induced hypoglycaemia. We analysed metabolites of hepatic glucose production under non-stressed conditions and after stress in comparison to wildtype controls. Analysis included glycogen, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), glycerol-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP). We also measured the activity of the key enzyme glucose-6-phosphatase. Blood and liver glucose were found to be low after stress, and liver glycogen was depleted. In addition, hepatic G6P and F6P were significantly reduced, especially during hypoglycaemia. Importantly, the activity of the enzyme converting G6P into glucose was not impaired. These data indicate a reduced rate of gluconeogenesis. The levels of DHAP and G3P were significantly lower suggesting decreased availability of glucose precursors from glycerol. This study gives biochemical evidence of impaired gluconeogenesis as one of the causes for hypoglycaemia observed in VLCAD deficiency. Whether this is due to lack of a substrate, inhibitory effects on other gluconeogenic enzymes or impaired transcription of gluconeogenic enzymes needs to be resolved in the future.

Autonomy or nonautonomy of gene effects in mosaics.

The dominant autosomal gene Msc (Multiple sex combs) of Drosophila causes the appearance of sex combs on the second and third legs and a reduction of size of sex combs on first legs. The effect of Msc was studied in mosaic flies in which non-Msc spots were present on a Msc background. Mosaics were due to somatic crossing-over induced by x-rays. Analysis of mosaics suggested autonomy of the action of Msc, provided that the somatic crossing-over occurs less frequently proximally to the Msc locus than distally. This unusual ratio of somatic crossing-over was confirmed by a separate experiment with the gene Kinked, whose locus is very close to Msc.