Proteome analysis of a rat liver nuclear insoluble protein fraction and localization of a novel protein, ISP36, to compartments in the interchromatin space

A rat liver nuclear insoluble protein fraction was analyzed to investigate candidate proteins participating in nuclear architecture formation. Proteins were subjected to two-dimensional separation by reversed-phase HPLC in 60% formic acid and SDS/PAGE. The method produced good resolution of insoluble proteins. One hundred and thirty-eight proteins were separated, and 28 of these were identified. The identified proteins included one novel protein, seven known nuclear proteins and 12 known nuclear matrix proteins. The novel 36 kDa protein was further investigated for its subnuclear localization. The human ortholog of the protein was expressed in Escherichia coli and antibodies were raised against the recombinant protein. Exclusive localization of the protein to the nuclear insoluble protein fraction was confirmed by cell fractionation followed by immunoblotting. Immunostaining of mouse C3H cells suggested that the 36 kDa protein was a constituent of an insoluble macromolecular complex spread throughout the interchromatin space of the nucleus. The protein was designated 'interchromatin space protein of 36 kDa', ISP36.     

MAP1B‐LC1 prevents autophagosome formation by linking syntaxin 17 to microtubules

In fed cells, syntaxin 17 (Stx17) is associated with microtubules at the endoplasmic reticulum–mitochondria interface and promotes mitochondrial fission by determining the localization and function of the mitochondrial fission factor Drp1. Upon starvation, Stx17 dissociates from microtubules and Drp1, and binds to Atg14L, a subunit of the phosphatidylinositol 3‐kinase complex, to facilitate phosphatidylinositol 3‐phosphate production and thereby autophagosome formation, but the mechanism underlying this phenomenon remains unknown. Here we identify MAP1B‐LC1 (microtubule‐associated protein 1B‐light chain 1) as a critical regulator of Stx17 function. Depletion of MAP1B‐LC1 causes Stx17‐dependent autophagosome accumulation even under nutrient‐rich conditions, whereas its overexpression blocks starvation‐induced autophagosome formation. MAP1B‐LC1 links microtubules and Stx17 in fed cells, and starvation causes the dephosphorylation of MAP1B‐LC1 at Thr217, allowing Stx17 to dissociate from MAP1B‐LC1 and bind to Atg14L. Our results reveal the mechanism by which Stx17 changes its binding partners in response to nutrient status.     

Degradation of huntingtin mediated by a hybrid molecule composed of IAP antagonist linked to phenyldiazenyl benzothiazole derivative

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by aggregation of mutant huntingtin (mHtt), and removal of mHtt is expected as a potential therapeutic option. We previously reported protein knockdown of Htt by using hybrid small molecules (Htt degraders) consisting of BE04, a ligand of ubiquitin ligase (E3), linked to probes for protein aggregates. Here, in order to examine the effect of changing the ligand, we synthesized a similar Htt degrader utilizing MV1, an antagonist of the inhibitor of apoptosis protein (IAP) family (a subgroup of ubiquitin E3 ligases), which is expected to have a higher affinity and specificity for IAP, as compared with BE04. The MV1-based hybrid successfully induced interaction between Htt aggregates and IAP, and reduced mHtt levels in living cells. Its mode of action was confirmed to be the same as that of the BE04-based hybrid. However, although the affinity of MV1 for IAP is greater than that of BE04, the efficacy of Htt degradation by the MV1-based molecule was lower, suggesting that linker length between the ligand and probe might be an important determinant of efficacy.     

Biological effects of Er:YAG laser irradiation on the proliferation of primary human gingival fibroblasts

We investigated the biological effects of Er:YAG laser (2940‐nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm^?2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm^?2 promoted maximal cell proliferation, determined by WST‐8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post‐irradiation. Elevation of ATP level, Ki67 staining, and cyclin‐A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post‐irradiation at 6.3 J cm^?2, and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up‐regulation of 21 genes involved in heat‐related biological responses and ER‐associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real‐time PCR. Surface temperature measurement confirmed that 6.3 J cm^?2 generated heat (40.9°C post‐irradiation). Treatment with 40°C‐warmed medium increased proliferation. Laser‐induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm^?2 strongly potentiated HGF proliferation via photo‐thermal stress, suggesting potential wound‐healing benefit.      

Structures and Chromosomal Localizations of the Glycosylphosphatidylinositol Synthesis GenePIGCand Its PseudogenePIGCP1

More than 10 genes are involved in the biosynthesis of glycosylphosphatidylinositol (GPI), which anchors many mammalian cell surface proteins to the membrane. Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in a GPI biosynthesis gene within the hematopoietic stem cell. The X-linked genePIGAhas been found to be mutated in all patients with PNH. This is probably because all other GPI synthesis genes are autosomal; hence two somatic mutations must occur to cause PNH, whereas one somatic mutation is sufficient to inactivatePIGA.Consistent with this notion, three other genes,PIGB, PIGF,andPIGH,are autosomal. Here we isolated a genomic clone of another GPI-synthesis gene,PIGC,and mapped it to chromosome 1q23–q25, further supporting this notion.PIGCis an intronless gene. We found an intronless pseudogene ofPIGC, PIGCP1,and mapped it to chromosome 11p12–p13. The presence of a processed pseudogene is a common feature ofPIGA, PIGF,andPIGC.     

Molecular Cloning of a Novel Vascular Endothelial Growth Factor, VEGF-D

We have identified and characterized a novel vascular endothelial growth factor (VEGF), VEGF-D, which is structurally related to vascular endothelial growth factor C. A full-length cDNA for human VEGF-D was cloned following the identification of an EST obtained through a TFASTA search of public EST databases. The murine VEGF-D was subsequently isolated from a mouse lung cDNA library. The human VEGF-D gene was mapped to human chromosome Xp22.31. Both human and mouse VEGF-D are strongly expressed in lung and encode the eight cysteine residues that are highly conserved among the members of this family. The high level of conservation between mouse and human VEGF-D may emphasize the biological importance of this gene. Recently the murine gene, FIGF, which is identical to mouse VEGF-D, was reported.     

Effect of lipids on insect cell growth and expression of recombinant proteins in serum-free medium

The lipid emulsion components of a serum-free insect cell medium were varied and evaluated for effects on cell growth and recombinant protein expression. The growth of High-Five^TM cells was significantly affected by polyol Pluronic F-68 and Tween-80, but not by lipids. Pluronic was essential for cell growth, while Tween-80 was required to achieve maximum cell densities. A dose response effect was observed for Tween-80 with optimal cell growth at a concentration of 25 mg/l. Cholesterol had a minor effect on cell growth, but was essential for the expression of recombinant proteins. The expression of -galactosidase (-gal) was directly affected by cholesterol with optimal expression at a concentration of 5.4 mg/l. Vitamin E, important as an antioxidant to stabilize lipids, did not directly affect recombinant protein expression. Although lipids were not required for cell growth, the presence of lipids were required during the cell growth phase in order to achieve efficient infection with baculovirus. These studies help to define the important components, and range of concentrations, for lipid emulsions which can effectively replace serum in insect cell culture.Abbreviations -gal galactosidase (E.C. 3.2.1.23) - Sf-9 Spodoptera frugiperda - High-5 Trichoplusia ni 5Bl-4     

Cloning of the Gene Encoding a Protochlorophyllide Reductase: the Physiological Significance of the Co-Existence of Light-Dependent and -Independent Protochlorophyllide Reduction Systems in the Cyanobacterium Plectonema boryanum

Cyanobacteria have two protochlorophyllide (Pchlide) reductasescatalyzing the conversion of Pchlide to chloro-phyllide, a keystep in the biosynthetic pathway of chlorophylls (Chls); a light-dependent(LPOR) and a light-independent (DPOR) reductase. We found anopen reading frame (ORF322) in a 2,131-bp EcoRI fragment fromthe genomic DNA of the cyanobacterium Plectonema boryanum. Becausethe deduced amino acid sequence showed a high similarity tothose of various plant LPORs and the LPOR activity was detectedin the soluble fraction of Esche-richia coli cells over-expressingthe ORF322 protein, ORF322 was defined as the por gene encodingLPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolatedby targeted mutagenesiss to investigate the physiological importanceof LPOR. YFP12 grew as well as wild type under low light conditions(10-25 µE m^–2 S^–1). However, its growth wassignificantly retarded as a result of a significant decreasein its Chl content under higher light conditions (85-130 µEm^–2 s^–1). Furthermore, YFP12 stopped growing andsuffered from photobleaching under the highest light intensity(170 µE m^–2 s^–1). In contrast, a chlL-dis-rupted(DPOR-less) mutant YFC2 grew as well as wild type irrespectiveof light intensity. From these phenotypic characteristics, weconcluded that, although both LPOR and DPOR contribute to Chlsynthesis in the cells growing in the light, the extent of thecontribution by LPOR increases with increasing light intensity;without it, the cells are unable to grow under light intensitiesof more than 130 µ Em^–2s-^–. (Received September 26, 1997; Accepted November 21, 1997)     

Origin and divergence of tandem repeats of primate D4 dopamine receptor genes

Human D4 dopamine receptor (D4DR) is polymorphic in terms of the repeat, numbers of the 48-base pairs (bp) sequence located in the third cytoplasmic loop of the receptor. The repeated sequence and its polymorphism in D4DR genes have also been identified in higher non-human primates, suggesting that the structure of D4DR has been maintained during primate evolution. To clarify the origin and divergence of the polymorphism in the D4DR gene, we determined the nucleotide sequence of this region of the D4DR gene in several species of prosimians and the tree shrew, a species which is closely related to primates. Prosimians except the tarsier had one or two unit(s) of the 48-bp sequence, and conserved sequences were recognized in most of the units of the prosimians. The tree shrew had only one unit of the 48-bp sequence, and its sequence was 71–75% identical to those of the nuits of galago, loris, and lemur. These findings suggest that the ancestral primate presumably had one 48-bp unit, and duplication of the unit occurred at the stage of prosimians. Tarsiers appeared to be distinct from other prosimians and simians because of the high repeat numbers of units and their sequences.