The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection

In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.     

Astragaloside IV attenuates orbital inflammation in Graves’ orbitopathy through suppression of autophagy

Introduction

Graves’ orbitopathy (GO) is an autoimmune inflammatory disorder affecting the orbit around the eye. Astragaloside IV (AS-VI) is the main active ingredient of the Chinese herbal medicine Huangqi (Radix Astragali Mongolici). AS-IV exhibits antioxidant and anti-inflammatory properties, and shows therapeutic potential in a number of ischemic and inflammatory diseases; however, its pharmaceutical activities in GO remain undefined.

Materials and methods

In this study, we investigated the effects of AS-IV on interleukin (IL)-1β-induced orbital fibroblast inflammation in vitro and GO orbital inflammation and ocular histopathological changes in vivo, as well as the underlying mechanisms responsible for these effects.

Results and conclusion

The results show that IL-1β increased mRNA expression of the inflammatory cytokines IL-6, IL-8, TNF-α, and MCP-1 in cultured orbital fibroblasts. This IL-1β-induced inflammation was accompanied by increased autophagic activity as reflected in increased Beclin-1 and Agt-5 expression, as well as LC3-I to LC3-II conversion. Pretreatment with the autophagy inhibitors 3-MA and bafilomycin A1, or silencing of autophagy-related proteins Beclin-1 and Atg-5, prevented IL-1β-induced orbital fibroblast inflammation, while pretreatment with the autophagy activator rapamycin had the opposite effects. These data suggested that autophagy was involved in GO orbital inflammation. AS-IV treatment significantly decreased IL-1β-induced inflammatory cytokine production in orbital fibroblasts in vitro and attenuated GO orbital inflammation, fat accumulation, collagen deposition, and macrophage infiltration in vivo. These in vitro and in vivo protective effects of AS-IV against GO were accompanied by decreased autophagic activities in orbital fibroblasts and GO orbital tissues, respectively. Collectively, our findings suggested that AS-IV protects against GO through suppression of autophagy. Thus, AS-IV may have preventive benefits for GO.

     

Nephritogenicity and α-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane

Nephritogenicity (anti-GBM-nephritis-inducing activity) and -chain composition of globular-domain (NC1) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different chains of type IV collagen. A purified nephritogenic fraction from renal BM contained 1–6(IV)NC1, whereas a non-nephritogenic fraction contained only 1–2(IV)NC1. Renal and pulmonary NC1 had strong nephritogenic activity; placental NC1 had weak activity. The renal and pulmonary fractions contained 1–6(IV)NC1, and the placental fraction had a large amount of 1–2(IV)NC1 and a very small amount of 3–6(IV)NC1. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained 1–5(IV) chains, but not the 6(IV) chain. The absence of 6(IV) chain in glomerular BM in bovine and in humans indicates that 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of 3–5(IV)NC1.     

Determinants of the differential gating properties of Cav3.1 and Cav3.3 T-type channels: a role of domain IV?

We have investigated the channel structural determinants that underlie the difference in gating properties of Cav3.1 and Cav3.3 T-type channels, by creating a series of chimeric channel constructs in which the major transmembrane domains were swapped. The chimeras were then expressed in tsA-201 cells and subjected to whole cell patch clamp analysis. Our data reveal that domains I and IV are major determinants of the half-activation potential. Substitution of domain IV was the most important determinant of activation time constant and time constant for recovery from inactivation, with domains I and II mediating a smaller role. In contrast, the carboxy terminal region did not appear to be involved. Determinants of the time constant for inactivation could not be localized to a specific transmembrane domain, but the concomitant substitution of domains I+IV was able to partially confer the inactivation kinetics among the two wild type channels. Our data indicate that the domain IV region mediates an important role in T-type channel activation, whereas multiple channel structural determinants appear to control T-type channel inactivation.     

Retinal Dystrophy and Optic Nerve Pathology in?the Mouse Model of Mucolipidosis IV

Mucolipidosis IV is a debilitating developmental lysosomal storage disorder characterized by severe neuromotor retardation and progressive loss of vision, leading to blindness by the second decade of life. Mucolipidosis IV is caused by loss-of-function mutations in the MCOLN1 gene, which encodes the transient receptor potential channel protein mucolipin-1. Ophthalmic pathology in patients includes corneal haze and progressive retinal and optic nerve atrophy. Herein, we report ocular pathology in Mcoln1^−/− mouse, a good phenotypic model of the disease. Early, but non-progressive, thinning of the photoreceptor layer, reduced levels of rhodopsin, disrupted rod outer segments, and widespread accumulation of the typical storage inclusion bodies were the major histological findings in the Mcoln1^−/− retina. Electroretinograms showed significantly decreased functional response (scotopic a- and b-wave amplitudes) in the Mcoln1^−/− mice. At the ultrastructural level, we observed formation of axonal spheroids and decreased density of axons in the optic nerve of the aged (6-month-old) Mcoln1^−/− mice, which indicates progressive axonal degeneration. Our data suggest that mucolipin-1 plays a role in postnatal development of photoreceptors and provides a set of outcome measures that can be used for ocular therapy development for mucolipidosis IV.Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by severe psychomotor retardation and visual loss. MLIV is classified as a lysosomal storage disease because of abnormal accumulation of storage material in lysosomes of all cells and tissues of the body.¹ Corneal clouding because of accumulation of lysosomal storage is an early pathological hallmark of the disease that, when present with developmental delay in infanthood, is highly suggestive of MLIV.Ophthalmic manifestations in patients generally have a progressive course and, in addition to corneal clouding, include optic nerve atrophy and outer retinal degeneration.^{2, 3, 4} In most of the patients, MLIV leads to blindness in the second decade of life.⁵Mutations in MCOLN1, which encodes the transient receptor potential cation channel TRPML1 (alias mucolipin-1) cause the disease.^{6, 7, 8, 9} More than 20 mutations in MCOLN1 have been identified to date.⁵ More than 75% of known MLIV patients are Ashkenazi Jewish, and the two founder mutations, present in 95% of Ashkenazi Jewish patients, result in complete loss of mRNA and protein.¹⁰ MLIV is a rare disease with carrier frequency of 1:100 in Ashkenazi Jewish and 1:10,000 in the general population. Many patients with MLIV remain undiagnosed or are misdiagnosed with cerebral palsy. Thus, bringing awareness of this disease to pediatric ophthalmologists and neurologists is important to improve diagnosis as new therapies are developed.Mucolipin-1 has six transmembrane domains and is permeable to Ca²⁺, Na⁺, K⁺, Fe²⁺, Mn²⁺, and Zn²⁺.^{11, 12, 13} Its channel activity is regulated by both calcium concentration and pH, and mucolipin-1 has been shown to have lipase activity.¹⁴ Previous studies by our group and others showed mucolipin-1 localization to the late endosomes and lysosomes.^{15, 16, 17, 18} The transient receptor potential channel protein mucolipin-1 is required for transport of lipids from the late endosomes-lysosomes to the trans-Golgi compartment,^{19, 20} Ca²⁺-dependent late endosome-lysosome fission-fusion events,²⁰ reformation of lysosomes from endosome-lysosome hybrids^{18, 21} and autolysosomes,^{22, 23} and lysosomal exocytosis.^{24, 25} Mucolipin-1 is strongly expressed in the mouse retina, with the highest mRNA levels in the outer plexiform layer and outer nuclear layer.²⁶The Mcoln1 knockout (KO) mouse model recapitulates the main features of the human disease, and is a good phenotypic platform for investigating MLIV disease mechanisms.^{27, 28, 29, 30} At the ultrastructural level, typical MLIV storage inclusions have been found in the brain during embryonic development.³¹ Histochemical analysis in the young adult (2 months of age) Mcoln1^−/− mice has shown pronounced glial activation, reduced myelination, and no neuronal loss in the cerebrum in the regions most affected by gliosis.²⁸In this study, we used Mcoln1^−/− mice to characterize the consequences of mucolipin-1 loss on retinal morphology, optic nerve myelination, and visual function in the course of the disease. Major manifestations of ophthalmic pathology in Mcoln1^−/− mice were as follows: non-progressive thinning of the photoreceptor layer; profound accumulation of storage inclusions throughout the retina and in the optic nerve, including formation of large axonal spheroids; axonal degeneration in the optic nerve in older mice; hypertrophied lysosomes in photoreceptors and other retinal cells; and reduced visual function.     

Up-regulation of Synaptotagmin IV within amyloid plaque-associated dystrophic neurons in Tg2576 mouse model of Alzheimer’s disease

Aim

To investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimer’s disease pathogenesis and to define the cell types containing increased levels of Syt IV in the β-amyloid plaque vicinity.

Methods

Syt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markers for astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronal specific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex.

Results

Western blot analysis showed higher Syt IV levels in Tg2576 mice cortex than in WT cortex. Syt IV was found only in neurons. In plaque vicinity, Syt IV was up-regulated in dystrophic neurons. The Syt IV signal was not up-regulated in the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions).

Conclusions

Syt IV up-regulation within dystrophic neurons probably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimer’s disease and is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.The main pathological hallmarks of Alzheimer’s disease (AD) are the formation of amyloid plaques, neurofibrillary tangles, dystrophic neurites, and sometimes activation of glial cells in the brain (1,2). In the vicinity of amyloid plaques, neurons undergo dramatic neuropathological changes including metabolic disturbances such as altered energy metabolism, dysfunction of vesicular trafficking, neurite breakage, and disruption of neuronal connections (3-8).Synaptotagmin IV (Syt IV) is a protein involved in the regulation of membrane trafficking in neurons and astrocytes (9,10). In hippocampal neurons, it regulates brain-derived neurotrophic factor release (11) and is involved in hippocampus-dependent memory and learning (12,13). In astrocytes, it is implicated in glutamate release (10). Recent data show that Syt IV plays an important role in neurodegenerative processes (14). Syt IV expression could be induced by seizures, drugs, and brain injury. Its changes have been shown in several animal models of neurodegeneration (Parkinson’s disease, brain ischemia, AD) (14-25). However, the exact role of Syt IV in neurodegeneration is unknown.Our previous study showed that the expression of Syt IV mRNA and its protein in the hippocampus and cortex of Tg2576 mouse model for AD was increased in the tissue surrounding β-amyloid plaques (14). It is not clear whether Syt IV is expressed in astrocytes (10,26,27) or/and in neurons (28,29), ie, whether it regulates the release of pro- or anti-inflammatory cytokines from β-amyloid associated astrocytes or is involved in neuronal vesicular pathogenesis (5,30). Therefore, the present study aimed to determine the type of cells in which Syt IV up-regulation occurs.     

Genetic elimination of α3(IV) collagen fails to rescue anti-collagen B cells

Organ deposition of autoantibodies against the noncollagenous-1 domain of the α3 chain of type IV collagen leads to severe kidney and lung injury in anti-glomerular basement membrane disease. The origin and regulation of these highly pathogenic autoantibodies remains unknown. Anti-α3(IV) collagen B lymphocytes are predicted to mature in vivo ignorant of target antigen because α3(IV) collagen expression is highly tissue restricted and pathogenic epitopes are cryptic. However, a recent analysis of an anti-α3(IV)NC1 collagen autoantibody transgenic mouse model revealed that developing B cells are rapidly silenced by deletion and editing in the bone marrow. To dissect the role of collagen as central tolerogen in this model, we determined B cell fate in autoantibody transgenic mice genetically lacking α3(IV) collagen. We found that absence of the tissue target autoantigen has little impact on the fate of anti-α3(IV)NC1 B cells. This implies a more complex regulatory mechanism for preventing anti-glomerular basement membrane disease than has been previously considered, including the possibility that a second antigen present in bone marrow engages and tolerizes anti-α3(IV)NC1 collagen B cells.     

Relevances of microfilament and microtubule to the adhesion properties of the HCC onto the Collagen IV/ Laminin coated surface

INTRODUCTION　　Tumorinvasionisclearlyanactiveprocessundergoingthreestages:attach-ment,migrationandmetastasis〔1〕.Infacts,tumorinvasioncouldbeconsideredady-namicadhesiveprocess,whichcontainedmuchcomplexandsubtlephysiologicalandbiochemicalinteractionsbetw…     

Biodegradable polymer - cisplatin(IV) conjugate as a pro-drug of cisplatin(II)

A Pt(IV) complex was covalently conjugated to a new biodegradable amphiphilic tri-block copolymer, MPEG-b-PCL-b-PLL, which contains pendant amino groups, to form a polymeric pro-drug of cisplatin(II), MPEG-b-PCL-b-PLL/Pt(IV). The conjugate was assembled into nano-micelles. The Pt(IV) complex, the polymer carrier and the conjugate were characterized systematically. In vitro release experiments showed that drug release from the polymer-Pt(IV) micelles follows an acid responsive and oxidation-reduction sensitive kinetics. HPLC-ICP-MS analysis revealed that cisplatin(II) can be released from the conjugate under an acidic plus a reductive condition which is available inside a cancerous cell. In vitro MTT assay demonstrated that the polymer-Pt(IV) micelles display higher cytotoxicity against SKOV-3 tumor cells than both cisplatin(II) and Pt(IV) complex. This enhanced cytotoxicity is attributed to effective internalization of the micelles by the cells via endocytosis mechanism, which was observed by fluorescence imaging and by direct determination of the platinum uptake by the cells. This polymer-Pt(IV) conjugate is a promising polymeric pro-drug of cisplatin in micellar form. It can protect the Pt(IV) complex against blood clearance. It can enter cancerous cells via endocytosis mechanism and then cisplatin(II) can be released. Therefore, this polymeric pro-drug of cisplatin is expected to find clinical applications in the future.     

Early venous manifestation of Ehlers–Danlos syndrome Type IV through a novel mutation in COL3A1

Ehlers–Danlos syndrome (EDS) leads to abnormalities in the synthesis of collagen and complications involving arterial vessels. We describe here a mutation in the intron 14 of the COL3A1 gene leading to EDS Type IV (EDS IV) associated with venous manifestations only. The patient, an 18-year-old male, suffered from truncal varicosity of the long saphenous vein on both sides. Conventional stripping surgery of the left saphenous vein revealed an extremely vulnerable ectatic superficial femoral vein. An inserted vein graft occluded, and venous thrombectomy was unsuccessful. A conservative anticoagulant and compression therapy finally succeeded. This is the first report describing EDS IV due to a mutation in intron 14 of the COL3A1 gene leading to venous manifestations without affecting arterial vessels at clinical presentation. Our findings imply that molecular genetic analysis should be considered in patients with unusual clinical presentation and that conservative therapy should be applied until a suspected clinical diagnosis has been secured.     

The metabolic syndrome of fructose-fed rats: effects of long-chain polyunsaturated ω3 and ω6 fatty acids. IV. D-glucose metabolism by isolated pancreatic islets

The major aim of the present study was to search for changes of D-glucose metabolism in isolated pancreatic islets possibly involved in the alteration of their secretory response to the hexose, as observed when comparing rats exposed for 8 weeks to diets containing either starch and sunflower oil or fructose and sunflower oil, as well as rats exposed to diets containing fructose, sunflower oil and either salmon oil or safflower oil. The substitution of starch by fructose in the diet affected unfavourably D-glucose phosphorylation by the isolated islets. In the fructose-fed rats, there was a close parallelism between D-[5-3H]glucose utilization and the dietary ω3/ω6 fatty acid ratio. There was little to distinguish, however, between the four groups of rats in terms of D-[U-1?C]glucose oxidation. The paired ratio between D-[U-1?C]glucose oxidation and D-[5-3H]glucose utilization, which always increased as the concentration of the hexose was raised from 2.8 to 8.3 and 16.7 mM, was tightly related, in the fructose-fed rats, to the HOMA index for insulin resistance.     

Familial co-segregation of Coffin–Lowry syndrome inherited from the mother and autosomal dominant Waardenburg type IV syndrome due to deletion of EDNRB inherited from the father

We report an African–American family that was identified after the proposita was referred for diagnostic evaluation at 4½ months with a history of Hirschsprung and dysmorphic features typical of Waardenburg syndrome (WS). Family evaluation revealed that the father had heterochromidia irides and hypertelorism supporting the clinical diagnosis of WS; however, examination of the mother revealed characteristic facial and digital features of Coffin–Lowry syndrome (CLS). Molecular testing of the mother identified a novel 2 bp deletion (c.865_866delCA) in codon 289 of RPS6KA3 leading to a frame-shift and premature termination of translation 5 codons downstream (NM_004586.2:p.Gln289ValfsX5). This deletion also was identified in the proposita and her three sisters with a clinical suspicion of CLS, all of whom as carriers for this X-linked disorder had very subtle manifestations. The molecular confirmation of WS type 4 (Shah-Waardenburg; WS4) was not as straightforward. To evaluate WS types 1–4, multiple sequential molecular tests were requested, including Sanger sequencing of all exons, and deletion/duplication analysis using MLPA for PAX3, MITF, SOX10, EDN3 and EDNRB. Although sequencing did not identify any disease causing variants, MLPA identified a heterozygous deletion of the entire EDNRB in the father. This deletion was also found in the proposita and the oldest child. Since the heterozygous deletion was the only change identified in EDNRB, this family represents one of the few cases of an autosomal dominant inheritance of WS4 involving the endothelin pathway. Altogether, clinical evaluation of the family revealed one child to be positive for WS4 and two positive for CLS, while two children were positive for both diseases simultaneously (including the proposita) while another pair test negative for either disease. This kinship is an example of the coincidence of two conditions co-segregating in one family, with variable phenotypes requiring molecular testing to confirm the clinical diagnoses.     

检测IV型胶原蛋白双mAb夹心ELISA的建立

慢性肝病通常发展为肝纤维化 ,常规实验室检查对早期肝纤维化的诊断敏感性差 ,且特异性不强。因此 ,建立一种可定量分析肝纤维化的方法尤为重要。血清Ⅳ型胶原蛋白(ＣＯＬⅣ )的水平与肝纤维化的程度密切相关 ,并可作为肝纤维化早期诊断指标之一[1,2 ] 。因此 ,我们采用两株抗ＣＯＬⅣ的单克隆抗体 (ｍＡｂ) ,建立了一种特异性强、灵敏度高、操作简便 ,适于临床检测血清ＣＯＬⅣ水平的双ｍＡｂ夹心ＥＬＩＳＡ法1　材料和方法1.1　材料　血清样品 4 9份 ,30份来自经体检排除心、肝、肾疾病的志愿者 (男女各半 ,年龄 2 6～ 5 0岁 ) ;19份采自…