Changes in the Carboxyl Terminus of the β Subunit of Human Propionyl-CoA Carboxylase Affect the Oligomer Assembly and Catalysis: Expression and Characterization of Seven Patient-Derived Mutant Forms of PCC in Escherichia coli

Propionyl-CoA carboxylase (PCC) catalyzes the biotin-dependent carboxylation of propionyl-CoA to -methylmalonyl-CoA in the mitochondrial matrix. Human PCC is a dodecamer composed of pairs of nonidentical α and β subunits encoded by PCCA and PCCB genes, respectively. Deficiency of PCC results in propionic acidemia (PA), a metabolic disorder characterized by severe metabolic ketoacidosis, vomiting, lethargy, and hypotonia. To date, almost 60 mutations have been reported in both genes. Exon 15 of the β subunit is one of the two sites where a number of mutations have been identified in PA patients. In the primary βPCC sequence, these mutations lead to three substitutions (R512C, L519P, and N536D), three truncations (R499X, R514X, and W531X), and one insertion (A51_R514insP). We expressed these mutant proteins in Escherichia coli in which the GroESL complex was overexpressed. The only mutation that does not impact the stability of mutant βPCC in bacteria is W531X. The remaining mutations lead to either complete (L519P, N536D) or partial (R499X, R512C, A513_R514insP, and R514X) degradation of the mutant subunits. Size-exclusion chromatography revealed that R512C and W531X do not affect the assembly of αPCC and βPCC to active oligomers. Specific activities for these mutant proteins, however, were only 3.9 and 10% of the wild type, respectively. Taken together, the carboxyl-terminal portion of 40 amino acid residues of the β subunit affects the stability and the assembly of the α and β subunits as well as the carboxylation of propionyl-CoA.     

Molecular and phenotypic characteristics of seven novel mutations causing branched‐chain organic acidurias

Specific mitochondrial enzymatic deficiencies in the catabolism of branched‐chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease‐causing mutations were identified in nine unrelated branched‐chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non‐responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South‐Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype–phenotype correlation for these rare diseases.     

Effect of molecular weight and polydispersity on kinetics of dissolution and release from pH/temperature-sensitive polymers

N-isopropylacrylamide (NIPAAm) polymers exhibit a lower critical solution temperature (LCST). Aqueous solutions of these polymers are soluble below their LCST and precipitate above their LCST. The LCST is dependent on pH for polymers with ionizable groups because of a change in hydrophilicity with ionization and electrostatic repulsion that cause a shift in the LCST. We have designed a novel polymeric delivery system that utilizes linear, pH/temperature-sensitive terpolymers of NIPAAm, butyl methacrylate (BMA) and acrylic acid (AA). This system allows the aqueous loading of drugs in polymeric beads with high loading efficiency while preserving the bioactivity of the protein drug. Furthermore, the unique properties of the pH/temperature-sensitive polymeric bead make it a potential system for oral drug delivery of peptide and protein drugs to different regions of the intestinal tract. This study aims at investigating the effect of polydispersity and molecular weight (MW) of terpolymers of poly(NIPAAm-co-BMA-co-AA) with feed mol ratio of NIPAAm/BMA/AA 85/5/ 10 on the polymer dissolution rate and on the release kinetics of a model protein, namely insulin. Varying the weight average MW (M_w) and polydispersity of the polymer modulated the polymer dissolution rate and the release rate of insulin from pH/temperature-sensitive polymeric beads. An increase in the polydispersity of the polymer through the addition of high MW polymer chains caused a decrease in the release rate of insulin and in the polymer dissolution rate. High MW polymer chains impose a certain degree of interaction between polymer chains due to chain entanglement. There is a limiting value of MW above which chain entanglement has no effect on drug release rate.     

Effect of Macromolecular Crowders on the Self‐Assembly Process of Silk Fibroin

Macromolecular crowding is known to have a significant impact on the aggregation and folding of proteins. In this study, the role of different macromolecular crowders such as polyethylene glycol (PEG 200 and PEG 6000) and dextran (dextran 6000 and 70000) on the self‐assembly of silk fibroin protein is explored. The data show that crowders induce a clear transition from random coil to β‐sheet secondary conformation in the fibroin as demonstrated by ThT fluorescence spectroscopy, circular dichroism, and ATR‐FTIR findings. However, this transition is not observed in case of control sample (fibroin without crowders). Excluded volume effects accelerate the kinetics of self‐association of the fibroin protein. Increasing the molecular weight of crowders increases the required time for maximum aggregation. This study suggests that treating the fibroin with different macromolecular crowders has modulated the propensity of fibroin going into β‐sheet conformation.     

Assembly Properties of an Alanine-Rich, Lysine-Containing Peptide and the Formation of Peptide/Polymer Hybrid Hydrogels

An alanine‐based peptide doped with charged lysines with a sequence of (AKA₃KA)₂ (AK2) was selected from the crosslinking regions of the natural elastin as a starting point for the fabrication of chemically diverse and structurally complex peptide/polymer hybrid hydrogels. At low peptide concentration, Pluronic F127 (F127) micelles are capable of increasing the peptide helicity and stabilizing it against thermal denaturation. At higher peptide concentration in basic media, the AK2 peptide developed a substantial amount of β‐sheet structure. The self‐assembled nanoscale structures were covalently interlocked. The crosslinked hybrid hydrogels were viscoelastic, exhibiting an elastic modulus of approximately 17 kPa and a loss tangent of 0.2.

     

Mutation Screening of the BRCA1 Gene in Early Onset and Familial Breast/Ovarian Cancer in Moroccan Population

Worldwide variation in the distribution of BRCA mutations is well recognised, and for the Moroccan population no comprehensive studies about BRCA mutation spectra or frequencies have been published. We therefore performed mutation analysis of the BRCA1 gene in 121 Moroccan women diagnosed with breast cancer. All cases completed epidemiology and family history questionnaires and provided a DNA sample for BRCA testing. Mutation analysis was performed by direct DNA sequencing of all coding exons and flanking intron sequences of the BRCA1 gene. 31.6 % (6/19) of familial cases and 1 % (1/102) of early-onset sporadic (< 45 years) were found to be associated with BRCA1 mutations. The pathogenic mutations included two frame-shift mutations (c.798_799delTT, c.1016dupA), one missense mutation (c.5095C>T), and one nonsense mutation (c.4942A>T). The c.798_799delTT mutation was also observed in Algerian and Tunisian BC families, suggesting the first non-Jewish founder mutation to be described in Northern Africa. In addition, ten different unclassified variants were detected in BRCA1, none of which were predicted to affect splicing. Most unclassified variants were placed in Align-GVGD classes suggesting neutrality. c.5117G>C involves a highly conserved amino acid suggestive of interfering with function (Align-GVGD class C55), but has been observed in conjunction with a deleterious mutation in a Tunisian family. These findings reflect the genetic heterogeneity of the Moroccan population and are relevant to genetic counselling and clinical management. The role of BRCA2 in BC is also under study.     

Effect of epitalon on the lifespan increase in Drosophila melanogaster

The geroprotector activity of epitalon, a synthetic tetrapeptide Ala–Glu–Asp–Gly, was studied on the Drosophila melanogaster wild strain Canton-S. The substance was added to the culture medium only at the developmental stage (from egg to larva). Epitalon significantly increased the lifespan (LS) of imagoes by 11–16% when applied at unprecedented low concentrations — from 0.001×10^–6 to 5×10^–6 wt.% of culture medium for males and from 0.01×10^–6 to 0.1×10^–6 wt.% of culture medium for females. The increase in LS did not depend on the substance dose. Effective concentrations of epitalon were 16 000–80 000 000 times lower than those of melatonin. The possible mechanisms of the antioxidant and regulatory effects of epitalon are discussed.     

MTS1／p16抑制基因的克隆及其对宫颈癌细胞系的影响

利用重组ＲＴ－ＰＣＲ方法从人胎盘中扩增多重肿瘤抑制基因（ＭＴＳ１）全长ｃＤＮＡ片断，克隆测序后，亚顾隆入哺乳动物高铲表达质粒ｐＣＥＰ中。将表达质业转染两种遗传背影不同的吕颈癌细胞系，发现外源基因ＭＴＳ１／ｐ１６基因的导入对ＨＰ〖Ｖ阳性的宫颈癌细胞系具有明显生长抑制作用，并出现细胞滞留Ｇ１期的特性。     

The Effect of Immunoglobulin Occurring on Human Sperm In Vivo on the Human Sperm/Hamster Ova Penetration Assay

ABSTRACT: We selected 91 infertile men who were tested for increased sperm-associated immunoglobulin and also tested in the human sperm/hamster ova penetration assay. There was a statistically significant association between the presence of increased sperm-associated IgG alone (p = 0.0218) and both sperm-associated IgG and A (p = 0.0187) when correlated with the failure to penetrate any hamster ova. There was a trend but no statistical significance when sperm-associated immunoglobulin A alone was present. There was a trend but no statistical relationship between the presence of sperm-associated immunoglobulin and the sperm penetration assay when the criteria for normality of the sperm penetration assay was a 15% or greater ovum penetration rate.     

Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370

Antibacterial drugs in the nitrofuran series, such as nitrofurazone, furazidin, nitrofurantoin and nifuroxazide, as well as the nitric oxide generators sodium nitroprusside and isosorbide mononitrate in concentrations that do not suppress bacterial growth, were shown to increase the capacity of pathogenic bacteria Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370 to form biofilms. At 25–100 μg/ml, nitrofurans 2–2.5-fold enhanced biofilm formation of P. aeruginosa PAO1, and NO donors 3–6-fold. For B. cenocepacia 370, the enhancement was 2–5-fold (nitrofurans) and 4.5-fold (sodium nitroprusside), respectively.     

Effect of Bordetella pertussis extract and vasoactive amines on vascular permeability

Vasxular permeability to Evans blue dye and 131-I-labeled human serum albumin was studied in normal mice and in mice treated with alkaline saline extracts (SE) from Bordetella pertussis cells. Skin sites inoculated intracutaneously with small doses of histamine, serotonin, or a combination of these 2 substances were more permeable in SE-treated mice than in normal animals. Intravenously administered catecholamines were able to reduce in varying degrees the vascular permeability induced by serotonin or by histamine in normal mice; in SE-treated mice the catecholamines were less effective. The relative effectiveness of intravenously administered catecholamines to reduce vascular permeability in normal or SE-treated mice was ranked as follows: isoproterenol greater than epinephrine greater than norepinephrine. When catecholamines were given concomitantly with histamine and serotonin in the skin test site, the permeability in both normal and SE-treated mice was again reduced or blocked, but isoproterenol was only weakly effective in this instance. Their relative effectiveness was epinephrine greater than norepinephrine greater than isoproterenol. The possible explanations for these results are discussed.     

Effects of Trifolium pratense and Cimicifuga racemosa on the endometrium of wistar rats

Objective

The aim of this study was to evaluate the effects of Trifolium pratense and Cimicifuga racemosa upon the endometrium of castrated female Wistar rats, comparing these results with a placebo and estradiol valerate.

Methods

Thirty-two adult castrated female Wistar rats were divided into four groups (eight rats per group) that receiving either tap water, estradiol valerate, isoflavones from T. pratense or deoxyactein from C. racemosa daily. The doses used were equivalent to normal doses used in humans. The results were analyzed by endometrial histology and the expression of α-estrogen receptor and protein Ki67. Both α-receptor and Ki67 expressions were determined by immunohistochemical and morphometric analysis.

Results

Endometrium histology stayed atrophic with both herbal extracts, but T. pratense supplementation increased the expression of α-estrogen receptors when compared to the placebo group, without protein Ki67 expression enhancement. Both herbal extracts presented a lower Ki67 expression when compared to the placebo group.

Conclusion

T. pratense presented α-estrogen receptor stimulation in the endometrium without increasing cell proliferation. Both herbal extracts reduced endometrial proliferation in comparison to the placebo group.     

Could beaked whales get the bends?: Effect of diving behaviour and physiology on modelled gas exchange for three species: Ziphius cavirostris, Mesoplodon densirostris and Hyperoodon ampullatus

A mathematical model, based on current knowledge of gas exchange and physiology of marine mammals, was used to predict blood and tissue tension N₂ (P_N2) using field data from three beaked whale species: northern bottlenose whales, Cuvier's beaked whales, and Blainville's beaked whales. The objective was to determine if physiology (body mass, diving lung volume, dive response) or dive behaviour (dive depth and duration, changes in ascent rate, diel behaviour) would lead to differences in P_N2 levels and thereby decompression sickness (DCS) risk between species. Diving lung volume and extent of the dive response had a large effect on end-dive P_N2. The dive profile had a larger influence on end-dive P_N2 than body mass differences between species. Despite diel changes in dive behaviour, P_N2 levels showed no consistent trend. Model output suggested that all three species live with tissue P_N2 levels that would cause a significant proportion of DCS cases in terrestrial mammals. Cuvier's beaked whale diving behaviour appears to put them at higher risk than the other species, which may explain their prevalence in strandings after the use of mid-frequency sonar.     

Effect of targeted disruption of signal transducer and activator of transcription (Stat)4 and Stat6 genes on the autoimmune diabetes development induced by multiple low doses of streptozotocin

The MLDS (multiple low doses of streptozotocin) model of diabetes was induced in Stat4^−/−, Stat6^−/−, and double-deficient Stat4^−/−/6^−/− mice to examine the role of STAT4/STAT6 deficiency in development of autoimmune diabetes. Cytokine production of T-cells from Stat4^−/− mice confirmed a predominantly Th2-type immune response. Stat4^−/− mice exhibited delayed onset and reduced severity of disease compared to wild-type (WT) mice. In contrast, STAT6 deficiency, with a predominant Th1 response, did not influence the kinetics or severity of MLDS-induced autoimmune diabetes. Interestingly, Stat4^−/−/6^−/− mice, with a prominent Th1-type response, experienced an accelerated and aggravated course of diabetes after MLDS, implicating a STAT4-independent Th1 response in the immunopathogenesis of MLDS-induced autoimmune diabetes. The sensitivity of islet cells from Stat4^−/− or Stat4^−/−/6^−/− mice to cytokines and STZ was not different from that of islet cells of WT mice. Hence, the observed effects of STAT4 and STAT4/6 deficiency on MLDS-induced autoimmune diabetes are likely due to their effects on T-cell responses.     

pH值对LAK细胞增殖的影响及与rIL-2联合抗恶性肿瘤的研究

目的 :调节细胞培养液 pH值 ,观察不同 pH值对LAK细胞增殖和rIL 2、LAK细胞杀伤恶性肿瘤细胞的影响。方法 :采用淋巴细胞分离液梯度离心法从正常人外周血分离单个核细胞并用rIL 2激活 ,于 96孔细胞培养板或培养管中培养。观察pH值分别为 6.8、7.3、7.6的培养液对rIL 2激活单个核细胞的影响。用 740 4细胞作为靶细胞 ,MTT法测定培养液 pH值为 6.8、7.3、7.6状态下rIL 2的杀伤作用和LAK细胞的细胞毒性。结果 :培养液 pH值为 7.3、7.6,有利于LAK细胞增殖 ,pH值为 7.6时LAK细胞杀伤效果最佳 ,pH值为 6.8时几乎无杀伤作用。培养液 pH值为 6.8、7.6都不利于740 4细胞生长 ,而 pH值为 7.6有利于LAK细胞生长。 结论 :恶性肿瘤组织内 pH值的变化与免疫细胞和肿瘤细胞的接触反应存在联系。     

“Low-risk” and “High-risk” HPV-infection and K-ras Gene Point Mutations in Human Cervical Cancer: A Study of 31 Cases

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for “low-risk” (types 6 and 11) and “high-risk” (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. “Low-risk” HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of “high-risk” human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that “high-risk” HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.     

Effect of saccharides on plasma mediated haemagglutination and in vitro phagocytosis by haemocytes of the snail Lymnaea stagnalis

To study the role of lectins in the defence system of the snailLymnaea stagnalis, saccharides were tested for their capacity to inhibit plasma-mediated haemagglutination of trypsinised and fixed mouse red blood cells (M-rbc) and fixed rabbit red blood cells (R-rbc).d-Arabinose,N-acetylneuraminic acid, fucoidan andd-melezitose were good agglutination inhibitors using both types of rbc.d-Fructose, however, stimulated haemagglutination. Based on these results, in vitro phagocytosis assays were performed withd-arabinose (an agglutination inhibitor) andd-fructose (an agglutination stimulator). In co-incubation experiments both sugars inhibited adherence and phagocytosis of M-rbc in a concentration-dependent manner. Adherence and phagocytosis of R-rbc was stimulated at the lowest concentrations of D-arabinose and most concentrations ofd-fructose. Preincubation experiments showed decreased adherence and phagocytosis after preincubation of M-rbc with either sugar. Adherence and phagocytosis of R-rbc was reduced after preincubation of haemocytes withd-fructose. Preincubation of R-rbc had little effect. Our results support former studies indicating the presence of lectins both in the snail plasma and on the haemocytes.     

环境与基因对边缘性人格障碍的影响

边缘性人格障碍(BPD)是精神科常见的一种人格障碍,虽然目前对BPD的发病机制尚未完全明了,但已有研究提示基因与环境及两者的交互作用在BPD的发生发展中具有非常重要的作用。本文通过CNKI,MEDLINE和PsysNET等数据库上检索有关边缘性人格与环境、基因之间关系的研究文献,并对文献进行系统总结,发现目前已有较多研究结果支持了基因与环境之间的交互作用在BPD发生发展中的作用。然而,已有关于两者交互作用相关研究尚存在缺陷,需未来的研究进行完善。