HumanDCTN1: Genomic Structure and Evaluation as a Candidate for Alström Syndrome

The human dynactin 1 gene (DCTN1) is positioned on chromosome 2p13, the candidate region for various diseases including Alström syndrome, limb-girdle muscle dystrophy, and Miyoshi myopathy. Here, we report the exon–intron structure ofDCTN1along with characterization of the 5′ upstream sequence and alternative splice variants previously identified by Tokitoet al.(1996),Mol. Biol. Cell7: 1167–1180). Knowledge of the genomic structure ofDCTN1allowed us to design intronic primers necessary for analyzing mutations in families segregating for diseases linked to this gene. These primers were tested on a French Acadian kindred segregating for Alström syndrome. No mutations were observed within the coding region ofDCTN1in this family. However, the intronic primers should allow for the rapid amplification of the coding region for mutational analysis of additional Alström families and other diseases tightly linked to theDCTN1locus on chromosome 2p13.     

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca2+ (0.1 mM concentration) and differentiated cells grown in high Ca2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin alpha (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.     

Characterization of two forms of poly(ADP-ribose) glycohydrolase in guinea pig liver

A poly(ADP-ribose) glycohydrolase from guinea pig liver cytoplasm has been purified approximately 45,000-fold to apparent homogeneity. The cytoplasmic poly(ADP-ribose) glycohydrolase designated form II differed in several respects from the nuclear poly(ADP-ribose) glycohydrolase I (Mr = 75,500) previously purified from the same tissue (Tanuma et al., 1986a). The purified glycohydrolase II consists of a single polypeptide with Mr of 59,500 estimated by a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 57,000 was determined by gel permeation. Peptide analysis of partial proteolytic degradation of glycohydrolases II and I with Staphylococcus aureus V8 protease revealed that the two enzymes were structurally different. Amino acid analysis showed that glycohydrolase II had a relatively low proportion of basic amino acid residues as compared with glycohydrolase I. Glycohydrolase II and I were acidic proteins with isoelectric points of 6.2 and 6.6, respectively. The optimum pH for glycohydrolases II and I were around 7.4 and 7.0, respectively. The Km value for (ADP-ribose)n (average chain length n = 15) and the Vmax for glycohydrolase II were 4.8 microM and 18 mumol of ADP-ribose released from (ADP-ribose)n.min-1.(mg of protein)-1, respectively. The Km was about 2.5 times higher, and Vmax 2 times lower, than those observed with glycohydrolase I. Unlike glycohydrolase I, glycohydrolase II was inhibited by monovalent salts. ADP-ribose and cAMP inhibited glycohydrolase II more strongly than glycohydrolase I. These results suggest that eukaryotic cells contain two distinct forms of poly(ADP-ribose) glycohydrolase exhibiting differences in properties and subcellular localization.     

Resonance Raman on the purple intermediates of the flavoenzyme D-amino acid oxidase

Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm^?1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm^?1 in the RR spectrum with D-alanine was shifted to 1675 cm^?1 upon [¹⁵N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm^?1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.     

Anion-induced conformational change of apo-electron-transferring flavoprotein.

Apoprotein of electron-transferring flavoprotein (ETF) exists in an equilibrium between two different forms, only one of which can associate with FAD (Sato, K. et al. (1991) J. Biochem. 109, 734-740), as represented in the following kinetic scheme: A* in equilibrium with A, A+FAD in equilibrium with holoETF, where "A*" and "A" are the different forms of apoETF. In the present study, the effects of various anions on the conversion between the two forms of apoETF were investigated by kinetic analyses on binding of FAD to apoETF. All the anions tested here induced the conversion from "A*" to "A"; the order of the effectiveness was I- approximately Br- greater than Cl- greater than F-. Further, glycerol also induced the conversion from "A*" to "A". The elution pattern of apoETF on molecular sieve chromatography was changed by addition of salts or glycerol; this change was due to the conversion from "A*" to "A" by the added solutes. The "A*" form was eluted more rapidly than the "A" form, indicating that the "A*" form exists in a looser conformation than the "A" form. The far-UV CD spectral change upon addition of salts indicated that a greater part of the secondary structure is retained in the conversion from "A*" to "A," but the "A" form contains a somewhat larger amount of beta-sheet than "A*."     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models

The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light scattering. The concentration (c) dependence of the apparent weight-average molecular weight (Mwapp) was determined over a wide concentration range of 0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme concentration, corresponded to the Mr value of the monomer. The self-association mode of the apoenzyme was systematically explored with nonlinear least-squares analysis of the Mwapp versus c data. The simplest model that fitted the data well was a model of isodesmic indefinite self-association of the monomer with the isodesmic association constant of 0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20 degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8, 3829-3838), did not fit the Mwapp versus c data either in the limited low concentration range or in the whole concentration range examined at 25 degrees C. To test the validity of the chosen model, the observed sedimentation boundary profiles were compared with the idealized boundary profiles calculated for the better-fit models. The profile calculated with the model of the isodesmic indefinite self-association mechanism was qualitatively consistent with the observed ones. The utility of the nonlinear least-squares procedure for analyzing self-associating systems was demonstrated.     

NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.     

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.     

Spatiotemporal variation in N2O flux within a slope in a Japanese cedar (Cryptomeria japonica) forest

Topographic factors affect nitrogen cycling in forest soils, including nitrous oxide (N₂O) emissions, which contribute to the greenhouse effect. We measured the N₂O flux at 14 chambers placed along a 65-m transect on a slope for 1 year at 2- to 3-week intervals. We applied a hierarchical Bayesian model with a conditional autoregressive (CAR) model to assess the spatiotemporal N₂O flux along a slope and quantify the effects of environmental factors on N₂O emissions. N₂O fluxes at chambers located at lower positions along the slope were relatively greater than those at higher positions. During the non-soil-freezing period, N₂O fluxes fluctuated seasonally depending on soil temperature. The soil temperature dependency of N₂O fluxes at each chamber increased with descending slope position (the median of the Q₁₀ equivalent simulated from posterior distribution ranged from 1.18 to 3.64). According to the Bayesian hierarchical model, this trend could be partially explained by the C/N ratio at each chamber position. During the soil-freezing period, relatively high N₂O fluxes were observed at lower positions along the slope.