Contribution of SNP arrays in diagnosis of deletion 2p11.2-p12

Deletions of the short arm of chromosome 2 are exceedingly rare, having been reported in few patients. Furthermore most cases with deletion in 2p11.2-p12 have been studied using standard karyotype and so it is not possible to delineate the precise size of deletions.Here, we describe a 9-year-old girl with a 9.4 Mb de novo interstitial deletion of region 2p11.2-p12 identified by SNP array analysis.The deleted region encompasses over 40 known genes, including LRRTM1, CTNNA2 and REEP1, haploinsufficiency of which could explain some clinical features of this patient such as mental retardation, speech delay and gait abnormalities.A comparison of our case with previously reported patients who present deletions in 2p11.2-p12 was carried out.Our case adds new information to the deletion of 2p11.2-p12, improving the knowledge on this rearrangement.     

Hsp31, a member of the DJ-1 superfamily,is a multitasking stress responder with chaperone activity

Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses.     

Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint

The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to Cdc20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). Cdc20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31(comet) (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31(comet) protein antagonizes the ability of Mad2 to inhibit APC/C(Cdc20) in vitro and in Xenopus egg extracts. Interestingly, p31(comet) binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31(comet) to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31(comet) forms a transient complex with APC/C(Cdc20)-bound Mad2. Purified p31(comet) enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2-Cdc20 interaction. Therefore, our results suggest that p31(comet) counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.     

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k_cat/K_m values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.     

人乳头瘤病毒HPV31型宫颈癌细胞模型的建立

宫颈癌与人乳头瘤病毒感染密切相关,建立宫颈癌实验模型可为宫颈癌的研究提供理想的模拟实验条件。我们通过应用基因重组技术,分别以HPV31型E6和E7基因为目的基因,通过原核表达、蛋白纯化和免疫小鼠等获得其特异性检测抗体。我们还通过构建E6和E7基因真核表达载体、转染C33A细胞、博莱霉素抗性筛选和表达检测等步骤,获得一种稳定的体外宫颈癌细胞系。经酶切鉴定及测序证实细胞基因组已重组插入质粒中的目的基因。我们已成功筛选到稳定的目的mRNA和蛋白表达的阳性细胞系,建立了稳定的人乳头状瘤病毒31型(HPV31)的宫颈癌细胞株,为研究宫颈癌提供了体外实验模型。     

Occurrence of A-kinase anchor protein and associated cAMP-dependent protein kinase in the inner compartment of mammalian mitochondria

Evidence showing the existence in the inner compartment of rat-heart mitochondria of AKAP121 and associated PKA is presented. Immunoblotting analysis and trypsin digestion pattern show that 90% or more of mitochondrial C-PKA, R-PKA and AKAP121 is localized in the inner mitochondrial compartment, when prepared both from isolated mitochondria or cardiomyocyte cultures. This localization is verified by measurement of the specific catalytic activity of PKA, radiolabelling of R-PKA by (32)P-phosphorylated C-PKA and of AKAP by (32)P-phosphorylated R-PKA and electron microscopy of mitochondria exposed to gold-conjugated AKAP121 antibody.     

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage-dependent anion-selective channels (VDACs) are pore-forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin-1. We swapped the VDAC3 N-terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N-terminus with the VDAC1 N-terminus caused the chimaera to become more active than VDAC1. The VDAC2 N-terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N-terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.     

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

The 970 loop (helix 31) of Escherichia coli 16S ribosomal RNA contains two modified nucleotides, m²G966 and m⁵C967. Positions A964, A969, and C970 are conserved among the Bacteria, Archaea, and Eukarya. The nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this ribosomal RNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Nonrandom nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m²G966 or m⁵C967 produce more protein in vivo than do wild-type ribosomes. Overexpression of initiation factor 3 specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for initiation factor 3 binding and for the proper initiation of protein synthesis.     

LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

Autologous adipose tissue is an ideal soft tissue filling material, and its biocompatibility is better than that of artificial tissue substitutes, foreign bodies and heterogeneous materials. Although autologous fat transplantation has many advantages, the low retention rate of adipose tissue limits its clinical application. Here, we identified a secretory glycoprotein, leucine‐rich‐alpha‐2‐glycoprotein 1 (LRG‐1), that could promote fat graft survival through RAB31‐mediated inhibition of hypoxia‐induced apoptosis. We showed that LRG‐1 injection significantly increased the maintenance of fat volume and weight compared with the control. In addition, higher fat integrity, more viable adipocytes and fewer apoptotic cells were observed in the LRG‐1‐treated groups. Furthermore, we discovered that LRG‐1 could reduce the ADSC apoptosis induced by hypoxic conditions. The mechanism underlying the LRG‐1‐mediated suppression of the ADSC apoptosis induced by hypoxia was mediated by the upregulation of RAB31 expression. Using LRG‐1 for fat grafts may prove to be clinically successful for increasing the retention rate of transplanted fat.     

First direct discrimination of chiral phosphine selenide (P=Se) derivatives by multinuclear magnetic resonance spectroscopy in the presence of a chiral dirhodium complex

Enantiomeric ratios of compounds with P=Se functionalities (phosphine selenides) can easily be determined by (1)H, (13)C, (31)P, and (77)Se NMR spectroscopic inspection of their diastereomeric complexes with (R)-Rh(2)(MTPA)(4) (MTPA-H identical with methoxytrifluoromethylphenylacetic acid; Mosher's acid). This is the first facile and rapid spectroscopic method for chiral recognition in this class of compounds. Whereas most complexation shifts Delta(delta) are moderate or even negligible, significant signal dispersions Delta(nu) can be observed. Some rationalization for the adduct formation mode is presented. NMR spectral characteristics of the free P=Se compounds 1-5 are described.     

白藜芦醇对小鼠溃疡性结肠炎的影响*

目的:研究白藜芦醇通过调节Wnt/β-catenin信号通路抗溃疡性结肠炎的作用机制。方法:①葡聚糖硫酸钠盐(DSS)诱发溃疡性结肠炎实验:28只C57BL/6小鼠随机分为4组(n=7):control组、 DSS组、DSS+白藜芦醇(DSS+Res)组和Res组。实验周期为3周,小鼠饮用DSS水诱导溃疡性结肠炎并给予白藜芦醇灌胃。实验期间每天称小鼠体重并观察小鼠活动和粪便情况。处理结束后,安乐死小鼠,取小鼠脾脏称重,取小鼠结肠测量长度。苏木精-伊红染色法(H&E)染色观察小鼠结肠组织病理改变;实时荧光定量PCR(qPCR)检测小鼠结肠组织miRNA-31的表达;Western Blot检测小鼠结肠组织β-catenin、Cyclin D1蛋白的表达。②离体实验:以10 mg/ml浓度的白藜芦醇处理HCT 116细胞,检测HCT 116细胞β-catenin、低密度脂蛋白受体相关蛋白6(LRP-6)、卷曲蛋白3(FZD3)、c-Myc蛋白的表达;HCT 116细胞转染miRNA-31 mimic和inhibitor,检测β-catenin蛋白的表达。结果:①DSS组小鼠实验期间体重下降明显,精神萎靡,活动减少,出现血便;处理结束后小鼠的结肠长度缩短,脾脏增大。而给予白藜芦醇后小鼠的以上情况得到改善。②白藜芦醇抑制了溃疡性结肠炎小鼠结肠组织miRNA-31的表达及β-catenin、Cyclin D1蛋白的表达。③白藜芦醇下调HCT 116细胞β-catenin、LRP-6、FZD3、c-Myc蛋白的表达。转染miRNA-31 inhibitor后,HCT 116细胞中β-catenin蛋白表达减少。结论:白藜芦醇能够抑制DSS诱导的小鼠溃疡性结肠炎,这种作用与下调Wnt信号通路有关,其对Wnt 信号的下调作用与miRNA-31有关。     

Limited proteolysis as a tool to study the kinetics of protein folding: Conformational rearrangements in acid-dissociated lactic dehydrogenase as determined by pepsin digestion

Noncovalent aggregation as a side reaction competing with the reconstitution of oligomeric enzymes is enhanced by slow conformational changes within the partially unfolded subunits. This has been shown for lactic dehydrogenase from pig muscle after acid dissociation [G., Zettlmeissl R. Rudolph, and R. Jaenicke (1981)Eur. J. Biochem.121, 169–175]. The present experiments confirm previous spectroscopic evidence (from circular dichroism) applying pepsin digestion and subsequent analysis of the fragments on sodium dodecyl sulfate-polyacrylamide gradient gels. The susceptibility of certain fragmentation sites toward pepsin digestion changes with increasing incubation at acid pH, in accordance with a slow M₁ → M₂ transition of the acid-dissociated monomers. Constant pulses of pepsin at varying times after transferring native enzyme to pH 2.3 yield distinct changes in the fragmentation pattern consisting of undigested monomers (M_r = 35,000) plus 12 fragments ranging from 31,000 to 5000. Short digestion of the M₂ species at low concentrations of pepsin preferentially yields 25,000 and 10,500 fragments (molar ratio pepsin:lactic dehydrogenase = 1:24). The time-dependent decrease of monomers upon incubation in 0.1 m sodium phosphate, pH 2.3, at 20 °C strictly parallels the formation of the two fragments. The quantitative kinetic analysis of the changes in peptide pattern yields a first-order rate constant K₁ = 8 ± 2 × 10^?4 s^?1. The observed increase in proteolytic susceptibility is in the time range of the above mentioned decrease in the far-ultraviolet circular dichroism, and the parallel decrease in the yield of reactivation. The results suggest that during the M₁ → M₂ transition at acid pH a specific interdomain cleavage site is becoming exposed. As taken from the molecular weight of the two main fragments the trp 225-lys 226 peptide bond is the most probable candidate for this cleavage site.     

Phosphorylation of the N-terminal portion of tyrosine hydroxylase triggers proteasomal digestion of the enzyme

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminal region of TH affects this stability. TH molecules phosphorylated at their Ser31 and Ser40 were localized predominantly in the cytoplasm of PC12D cells. However, those molecules phosphorylated at Ser19 were found mainly in the nucleus, whereas they seemed to be negligible in the cytoplasm. The inhibition of proteasomes increased the quantity of TH molecules phosphorylated at their Ser19 and Ser40, although it did not increase that of TH molecules or that of TH phosphorylated at its Ser31. The inhibition of autophagy did not affect the amount of the TH molecule or that of its three phosphorylated forms. Deletion mutants of human TH type-1 lacking the N-terminal region containing the three phosphorylation sites possessed high stability of the enzyme in PC12D cells. These results suggest that the phosphorylation of the N-terminal portion of TH regulates the degradation of this enzyme by the ubiquitin–proteasome pathway.     

氯化两面针碱对小鼠溃疡性结肠炎的干预作用及其机制*

目的:探讨氯化两面针碱(NC)通过靶向miR-31对葡聚糖硫酸钠(DSS)诱发小鼠结肠炎的保护作用及其机制。方法:用1%DSS诱发小鼠溃疡性结肠炎(UC)。30只雄性C57BL/6小鼠随机分为正常对照组(Control)(n=7),DSS组(n=8),DSS+NC组(7.27 mg/kg)(n=8)和NC组(n=7),饮水给予DSS,灌胃给予氯化两面针碱。造模周期为3周,分别为Control组和NC组每天饮用无菌水,DSS组和DSS+NC组第一周饮用1% DSS水,第2周正常饮水,第3周1% DSS水。造模最后一周给予Control组和DSS组小鼠0.5% 羧甲基纤维素钠(CMC-Na)灌胃,DSS+NC组和NC组给予NC灌胃。造模完成后,观察小鼠结肠炎相关的疾病活动指数(DAI),HE染色进行结肠组织病理评分,qPCR检测小鼠结肠组织miR-31的表达水平,Western blot检测小鼠结肠组织炎症蛋白NF-κB和COX-2的表达情况。结果:①与DSS组相比,DSS+NC组的 DAI 显著降低(P＜0.01),结肠病理损伤明显改善;②与Control组相比,DSS组小鼠结肠组织miR-31表达显著升高(P＜0.01),DSS+NC组miR-31的表达水平显著低于DSS组(P＜0.05);③与DSS组相比,DSS+NC组中的炎症蛋白NF-κB和COX-2表达水平显著下降(P＜0.05)。结论:氯化两面针碱对DSS诱导的小鼠溃疡性结肠炎有明显的治疗作用,其抗炎机制与下调miR-31的表达有关。     

First direct discrimination of chiral phosphorus thionate (P=S) derivatives by multinuclear magnetic resonance spectroscopy in the presence of a chiral dirhodium complex

Enantiomeric ratios of compounds with P=S functionalities can be determined by 1H, 13C, and 31P NMR spectroscopic inspection of their diastereomeric complexes with (R)-Rh2(MTPA)4 (MTPA-H identical with methoxytrifluoromethylphenylacetic acid; Mosher's acid). This is the first facile and rapid spectroscopic method for chiral recognition in this class of compounds. Whereas complexation shifts Deltadelta are moderate or even negligible, significant signal dispersions Delta(nu) can be observed. Some rationalization about the complexation mode is presented. The NMR spectral characteristics of the free P=S compounds 1-9 are described in detail.     

Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu²⁺-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies.     

Arabinogalactan protein 31 (AGP31), a putative network-forming protein in Arabidopsis thaliana cell walls?

Background and Aims

Arabinogalactan protein 31 (AGP31) is a remarkable plant cell-wall protein displaying a multi-domain organization unique in Arabidopsis thaliana: it comprises a predicted signal peptide (SP), a short AGP domain of seven amino acids, a His-stretch, a Pro-rich domain and a PAC (PRP-AGP containing Cys) domain. AGP31 displays different O-glycosylation patterns with arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on the Pro-rich domain. AGP31 has been identified as an abundant protein in cell walls of etiolated hypocotyls, but its function has not been investigated thus far. Literature data suggest that AGP31 may interact with cell-wall components. The purpose of the present study was to identify AGP31 partners to gain new insight into its function in cell walls.

Methods

Nitrocellulose membranes were prepared by spotting different polysaccharides, which were either obtained commercially or extracted from cell walls of Arabidopsis thaliana and Brachypodium distachyon. After validation of the arrays, in vitro interaction assays were carried out by probing the membranes with purified native AGP31 or recombinant PAC-V5-6xHis. In addition, dynamic light scattering (DLS) analyses were carried out on an AGP31 purified fraction.

Key Results

It was demonstrated that AGP31 interacts through its PAC domain with galactans that are branches of rhamnogalacturonan I. This is the first experimental evidence that a PAC domain, also found as an entire protein or a domain of AGP31 homologues, can bind carbohydrates. AGP31 was also found to bind methylesterified polygalacturonic acid, possibly through its His-stretch. Finally, AGP31 was able to interact with itself in vitro through its PAC domain. DLS data showed that AGP31 forms aggregates in solution, corroborating the hypothesis of an auto-assembly.

Conclusions

These results allow the proposal of a model of interactions of AGP31 with different cell-wall components, in which AGP31 participates in complex supra-molecular scaffolds. Such scaffolds could contribute to the strengthening of cell walls of quickly growing organs such as etiolated hypocotyls.     

Height, income, and nutrition in the Netherlands: the second half of the 19th century.

This paper explores the relationship between height and its determinants, paying explicit attention to the dynamic nature of the velocity of the growth profile. The relationship between height and some measures of income and nutrition is characterized by a changing lag pattern in 19th century the Netherlands.