Impact of Fabp1/Scp-2/Scp-x gene ablation (TKO) on hepatic phytol metabolism in mice

Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.     

The direction of human mesenchymal stem cells into the chondrogenic lineage is influenced by the features of hydrogel carriers

Low back pain is a major public health issue in the Western world, one main cause is believed to be intervertebral disc (IVD) degeneration. To halt/diminish IVD degeneration, cell therapy using different biomaterials e.g. hydrogels as cell carriers has been suggested. In this study, two different hydrogels were examined (in vitro) as potential cell carriers for human mesenchymal stem cells (hMSCs) intended for IVD transplantation. The aim was to investigate cell-survival and chondrogenic differentiation of hMSCs when cultured in hydrogels Puramatrix^® or Hydromatrix^® and potential effects of stimulation with growth hormone (GH). hMSCs/hydrogel cultures were investigated for cell-viability, attachment, gene expression of chondrogenic markers SOX9, COL2A1, ACAN and accumulation of extracellular matrix (ECM). In both hydrogel types, hMSCs were viable for 28 days, expressed integrin β1 which indicates adhesion of hMSCs. Differentiation was observed into chondrocyte-like cells, in a higher extent in hMSCs/Hydromatrix^® cultures when compared to hMSCs/Puramatrix^® hydrogel cultures. Gene expression analyses of chondrogenic markers verified results. hMSCs/hydrogel cultures stimulated with GH displayed no significant effects on chondrogenesis.In conclusion, both hydrogels, especially Hydromatrix^® was demonstrated as a promising cell carrier in vitro for hMSCs, when directed into chondrogenesis. This knowledge could be useful in biological approaches for regeneration of degenerated human IVDs.     

脑膜炎球菌P64K蛋白作为新型抗原载体的研究进展

P64K是来自奈瑟氏属脑膜炎球菌的一种外层膜蛋白,发现于脑膜炎恢复期患者和接种过古巴VAMENGOCBC疫苗的个体血清内。编码P64K蛋白的基因称lpdA,其编码序列长1782bp。P64K蛋白含593个氨基酸,由两个结构域组成,有二氢硫辛酸酰胺酰基脱氢酶活性。P64K是抗脑膜炎疫苗的候选者。因为P64K的高分子量和强免疫原性,被认为是一种很有前途的抗原载体 。     

应用端粒区带涂染探针检测染色体微小结构重排

为了评估染色体端粒区带涂染探针在遗传诊断的应用价值,应用显微切割获得的11q、12q和22q等3个染色体端粒区涂染探针（11q23.3→qter,12q24.1→qter,22q13.1→qter）,通过荧光原位杂交技术分析两个疑有染色体末端微小易位的习惯性流产病例。结果显示,病例1和病例2分别为t(11;12)和t(11;22)长臂末端间的微小易位,结合G显带技术确定断裂位点位于11q23.3、12q24.1、22q13.1。结果表明特异性染色体端粒区带探针可以确诊染色体末端区域的微小结构异常,可作为一种检出隐匿易位携带者并确定断裂位点的方法。     

Meeting report – Copper research at the top

In this brief paper, the author reports on a meeting on copper research (2nd International Meeting on Copper Homeostasis and its Disorders: Molecular and Cellular Aspects) recently held in Ravello, Italy (17–21 September 1999). Aimed at elucidating the diverse roles played by copper ions in biology and medicine, as they are currently intensely investigated worldwide, the meeting has been organized around a number of major topics from prominent areas of copper research. These included the molecular and cellular basis of copper transport, molecular advances in Menkes and Wilson's diseases, the involvement of copper in neurodegenerative diseases, the structure and function of copper metalloproteins.     

大鼠红细胞作为SOD新型载体的细胞水平上的研究

用低渗透析 -等渗重封的方法制备了包埋超氧化物歧化酶 ( SOD)的大鼠载体红细胞 ,并从细胞水平上研究了透析条件对大鼠红细胞包埋 SOD的影响与载体红细胞的部分性质 .流式细胞计( FCM)研究表明 ,随透析时间延长和透析液渗透压降低 ,包埋 SOD的载体红细胞百分率升高 ,但载体细胞平均包埋 SOD的量无明显变化 ;SOD浓度对载体细胞百分率无明显影响 ,但与载体细胞平均包埋 SOD的量成线性关系 ;载体红细胞前向角散射 ( FLS)明显下降 ,但显微镜下观察到的载体细胞的大小无明显变化 ,当载体细胞反注射到大鼠体内后 FLS能迅速恢复 ;载体红细胞密度下降 ,其原因是低渗透析时红细胞膨胀未能完全恢复 ;载体红细胞未暴露与自身 Ig G结合的抗原位点 .激光扫描共聚焦显微镜 ( LSCM)分析表明 ,SOD在细胞内呈从细胞中心到细胞膜浓度逐步下降的辐射分布特征 .     

Molecular mechanisms of copper metabolism and the role of the Menkes disease protein

Menkes disease is an X‐linked, recessive disorder of copper metabolism that occurs in approximately 1 in 200,000 live births. The condition is characterized by skeletal abnormalities, severe mental retardation, neurologic degeneration, and patient mortality in early childhood. The symptoms of Menkes disease result from a deficiency of serum copper and copper‐dependent enzymes. A candidate gene for the disease has been isolated and designated MNK. The MNK gene codes for a P‐type cation transporting ATPase, based on homology to known P‐type ATPases and in vitro experimentation. cDNA clones of MNK in Menkes patients show diminished or absented hybridization in northern blot experiments. The Menkes protein functions to export excess intracellular copper and activates upon Cu(I) binding to the six metal‐binding repeats in the amino‐terminal domain. The loss of Menkes protein activity blocks the export of dietary copper from the gastrointestinal tract and causes the copper deficiency associated with Menkes disease. Each of the Menkes protein amino‐terminal repeats contains a conserved ‐X‐Met‐X‐Cys‐X‐X‐Cys‐ motif (where X is any amino acid). These metal‐binding repeats are conserved in other cation exporting ATPases involved in metal metabolism and in proteins involved in cellular defense against heavy metals in both prokaryotes and eukaryotes. An overview of copper metabolism in humans and a discussion of our understanding of the molecular basis of cellular copper homeostasis is presented. This forms the basis for a discussion of Menkes disease and the protein deficit in this disease. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 93–106, 1999     

Carrier design: Synthesis and conformational studies of poly (L-lysine) based branched polypeptides with hydroxyl groups in the side chains

In the present study the development of a new series of branched polypeptides that contain hydroxyl groups in side chains is reported. Serine or threonine were attached by 1-hydroxy-benzotriazole catalyzed active ester method to the N-terminals of oligo (DL -alanine) chains grafted to a polylysine backbone resulted in poly[Lys-(Ser₁-DL -Ala_m)] (SAK) and poly-[Lys-(Thr_i-DL -Ala_m)] (TAK). Ser was coupled also directly to the η-amino groups of polylysine followed by polymerization of N-carboxy-DL -alanine anhydride resulting oligo (DL -Ala) chain terminals. In this way a reverse sequence was built up in the side chain corresponding to the poly[Lys-(DL -Ala_m-Ser_i)] (ASK). The number of hydroxyl groups in the polymer was increased by the synthesis of a branched polypeptide with oligo (DL -serine) branches instead of oligo (DL -alanine) ones—poly[Lys-(DL -Ser_m)] (SK). Classification of solution conformations of branched polypeptides was carried out by CD spectroscopy performed in water solution of various pH values and ionic strengths. Incorporation of single Ser residues in poly[Lys-(X_i)]-type polypeptides markedly promotes the formation of ordered structure without resulting precipitation even in high salt concentration. The presence of branches with multiple DL -Ser residues resulted in a slightly decreased ability of the polypeptide backbone to adopt an ordered conformation. Comparison of the CD properties of the SAK-ASK pair demonstrates that these compounds are similar, showing an increased tendency to form an ordered spatial arrangement in solution at elevated pH or ionic strength; however, differences in their CD spectra suggest that SAK has higher capability to form regular conformation under comparable conditions. The replacement of Ser by the Thr residue in poly[Lys-(X_i-DL -Ala_m)] induced a conformational transition and TAK exhibited a more helical structure. These results might indicate that not only hydrophobic or ionic attraction, but also H-bond interaction, can play a role in the formation and/or stabilization of ordered conformation of branched polypeptides. Findings with the hydroxyl group containing polymers reported in this paper can also explain their prolonged shelf stability and high water solubility. © 1997 John Wiley & Sons, Inc. Biopoly 42: 719–730, 1997     

A GX2GX3G motif facilitates acyl chain sequestration by Saccharomyces cerevisiae acyl carrier protein

Saccharomyces cerevisiae acyl carrier protein (ScACP) is a component of the large fungal fatty acid synthase I (FAS I) complex. ScACP comprises two subdomains: a conserved ACP domain that shares extensive structural homology with other ACPs and a unique structural domain. Unlike the metazoan type I ACP that does not sequester the acyl chain, ScACP can partially sequester the growing acyl chain within its hydrophobic core by a mechanism that remains elusive. Our studies on the acyl-ScACP intermediates disclose a unique ¹⁸⁸GX₂GX₃G¹⁹⁵ sequence in helix II important for ACP function. Complete loss of sequestration was observed upon mutation of the three glycines in this sequence to valine (G188V/G191V/G195V), while G191V and G188V/G191V double mutants displayed a faster rate of acyl chain hydrolysis. Likewise, mutation of Thr216 to Ala altered the size of the hydrophobic cavity, resulting in loss of C₁₂- chain sequestration. Combining NMR studies with insights from the crystal structure, we show that three glycines in helix II and a threonine in helix IV favor conformational change, which in turn generate space for acyl chain sequestration. Furthermore, we identified the primary hydrophobic cavity of ScACP, present between the carboxyl end of helix II and IV. The opening of the cavity lies between the second and third turns of helix II and loop II. Overall, the study highlights a novel role of the GX₂GX₃G motif in regulating acyl chain sequestration, vital for ScACP function.