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.

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.

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.

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.

[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.

[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.

[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.

Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress.

BACKGROUND: In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14-C18 acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and acylcarnitine profiles under stress. DESIGN: VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. RESULTS: Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14-C18 acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14-C18 acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 degrees C, the long-chain acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0.05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14-C18 acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. CONCLUSIONS: Long-chain acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.

[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.

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.

[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.

Tratamento da síndrome de Budd-Chiari com trombose de veia cava inferior por anastomose mesoatrial / Treatment of Budd-Chiari syndrome with venous cava thrombosis by mesoatrial shunt

A Síndrome de Budd-Chiari (SBC) é uma doença rara, ocorre com maior frequência nos adultos, não havendo predominância de sexo e é mais comum nos países do leste asiático. Relatamos o tratamento cirúrgico de paciente com SBC e trombose de veia cava. Paciente do sexo masculino, 29 anos, com Síndrome de Budd-Chiari consequente a trombose de veia cava inferior (VCI). Apresentava função hepática preservada, esplenomegalia, gastropatia congestiva com vários episódios de hemorragia digestiva alta e fígado com fibrose. Optou-se por realizar anastomose mesoatrial (AMA). Concluímos que AMA foi eficaz na descompressão hepática e resultou no desaparecimento dos sinais e sintomas da SBC além de ser seguida de melhora das provas de função hepática do paciente num seguimento de 8 meses

Distinct regulatory mechanism for p70 S6 kinase beta from that for p70 S6 kinase alpha.

BACKGROUND: A novel ribosomal S6 kinase, termed p70 S6 kinase beta (p70beta), has a highly homologous amino acid sequence to that of p70/p85 S6 kinase (p70alpha). This includes the critical phosphorylation sites, Thr252, Ser394 and Thr412 in p70alpha1, which correspond to Thr241, Ser383 and Thr401 in p70beta1, respectively. However, the regulatory mechanism for p70beta remains to be elucidated. RESULTS: We report here the expression and the mechanism of in vivo regulation of p70beta. Two isoforms, p70beta1 and p70beta2, were expressed in a variety of tissues at a different level. p70beta1 was mainly targeted to the nucleus, whereas p70beta2 dispersed throughout the cytoplasm including nucleoplasm. The kinase activity of p70beta1 was less sensitive to the inhibition induced by rapamycin, wortmannin and amino acid withdrawal than that of p70alpha. The portion of p70beta activity inhibited by rapamycin was rescued by the rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR). Mutational analysis revealed that the phosphorylation of Thr241 and Thr401 in p70beta1 was indispensable for the kinase activity. In contrast, a p70beta1 mutant in which Ser383 was substituted with Gly (S383G) still retained nearly the half maximal activity. Sequential phosphorylation of wild-type and S383G mutant of p70beta1 with mTOR and 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vitro synergistically activated their kinase activities. CONCLUSION: These results indicate that p70beta is regulated by the mTOR- and PDK1-signalling pathways through a synergistic interaction between phosphorylated Thr241 and Thr401, while Ser383 plays minor role in their activation mechanism. Activated p70beta may be less sensitive to dephosphorylation mediated by putative phosphatases activated by rapamycin, amino acid withdrawal, and probably wortmannin.

Carboxyl-terminal region conserved among phosphoinositide-kinase-related kinases is indispensable for mTOR function in vivo and in vitro.

BACKGROUND: The mammalian target of rapamycin (mTOR) belongs to the family of phosphoinositide (PI)-kinase-related kinases that includes the ataxia-telangiectasia gene product (ATM). mTOR plays a critical role in controlling translational effectors such as p70 S6 kinase alpha (p70 alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). RESULTS: We show that the C-terminal region of mTOR, which is highly conserved among the PI-kinase-related kinases, plays a critical role in the mTOR protein kinase activity. Deletion of the C-terminal residues did not adversely affect the expression of mTOR, but caused a nearly complete loss of the mTOR protein kinase activity toward both 4EBP1 and p70 alpha in vitro. These deletions also abolished the ability of a rapamycin-resistant mTOR mutant to rescue the activity of p70 alpha from inhibition induced by rapamycin in vivo. Furthermore, replacement of Trp2545, a conserved residue in the C-terminal region throughout the PI-kinase-related kinase family, abolished the function of the mTOR kinase, both in vivo and in vitro. However, substitution of 32 C-terminal residues of mTOR with those of ATM did not restore the mTOR function. CONCLUSIONS: These findings define an indispensable role for the noncatalytic C-terminal region of mTOR and indicate that, although this highly conserved region may be important throughout the PI-kinase-related kinase family, it is not functionally interchangeable within the family.

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.

Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro.

p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.

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.

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.

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.