鸡软骨肽化学结构特征及抗氧化、抗炎活性成分研究

目的:明确鸡软骨肽的主要化学结构特征,及其抗氧化、抗炎的物质基础。方法:对鸡软骨肽进行水解,采用异硫氰酸苯酯(PITC)-柱前衍生高效液相色谱法检测鸡软骨肽水解后所得氨基酸的组成;借助LC-MS/MS以及物种蛋白质数据库,确定鸡软骨肽中主要水解多肽的氨基酸序列;利用固相合成方法制备已知氨基酸序列的多肽,采用DPPH法、水杨酸法、FRAP方法测定合成肽段的抗氧化作用,采用H_2O_2致C518大鼠膝关节软骨细胞损伤模型测定合成肽段对氧化损伤的保护作用,采用LPS诱导的C518大鼠膝关节软骨细胞炎症模型测定合成肽段的抗炎作用。结果:确定18种氨基酸在鸡软骨肽中的占比情况,其中占比最高的是甘氨酸(G)29.5%,其次为脯氨酸(P)11.63%、丙氨酸(A)10.61%、谷氨酸(E)9.2%;从水解鸡软骨肽中鉴定出的11条分子量为794～1 860 Da的肽段,并筛选出多条抗氧化、抗炎作用显著的活性肽段,其中综合功效最佳的肽段为G4(MEGNAEESTLFCFAVRG)。结论:LC-MS/MS结合物种蛋白质数据库是一种快速、有效研究鸡软骨肽化学结构特征的新方法,本实验借助该方法初步确定了鸡软骨肽的化学结构特征以及抗氧化、抗炎活性成分,将为深入开展鸡软骨肽的应用研究提供基础。     

吗啡备用根大鼠脊髓组织神经营养活性物质的分离纯化和生物活性测定

目的　分离纯化吗啡备用根大鼠脊髓组织提取液 ,以期获得某些神经营养活性物质。　方法　应用SephacrylS 2 0 0HR凝胶层析、高效液相色谱 (HPLC)和组织培养等技术分离和检测神经营养活性物质。　结果　备用根大鼠脊髓组织提取液能够促进体外培养的鸡胚背根节 (DRG)神经突起的生长 ;吗啡作用的大鼠脊髓组织提取液也具有同样的作用 ,但备用根大鼠和吗啡作用的大鼠脊髓组织提取液在促神经突起生长作用方面并没有明显的差异。吗啡备用根大鼠脊髓组织提取液具有明显的神经营养活性作用。吗啡备用根大鼠脊髓组织提取液的SephacrylS 2 0 0HR凝胶层析Ⅱ峰洗脱液和Ⅳ峰洗脱液能够促进DRG神经突起的生长。经SDS PAGE分析 ,Ⅱ峰洗脱液呈现 1条分子量约为 6 5kD的蛋白质主带 ,Ⅳ峰洗脱液的蛋白质成分较为复杂。应用HPLC对凝胶层析Ⅳ峰洗脱液作进一步的分离 ,发现HPLCA峰洗脱液能够促进DRG神经突起的生长。经SDS PAGE显示 ,A峰洗脱液的两条蛋白质主带分子量分别为 30kD和 18kD。　结论　吗啡备用根大鼠脊髓组织提取液中具有神经营养活性作用的物质可能是分子量约为 6 5kD、30kD和 18kD蛋白质     

备用根大鼠脊髓后角组织神经营养活性物质的分离纯化和生物活性测定

目的　进一步分离纯化备用根大鼠部分去传入纤维支配 (手术侧 )的脊髓后角组织提取液 ,以获得某种神经营养活性物质。　方法　用 Superdex G- 75 prep grade凝胶层析、高效液相色谱 (HPL C)层析和细胞培养等技术分离和检测神经营养活性物质。　结果　手术侧脊髓后角组织提取液经 Superdex G- 75 prep grade凝胶层析和 HPL C层析后 ,得到的 A峰洗脱液具有促进体外培养鸡胚背根节 (DRG)分离神经元存活及其神经突起生长的神经营养活性作用。经 SDS-聚丙烯酰胺凝胶电泳 (PAGE)显示 ,A峰洗脱液呈现一条分子量约为 6 0 k D的蛋白质主带。　结论　结合生物活性检测结果 ,分子量约 6 0 k D的蛋白质可能是手术侧脊髓后角组织的神经营养活性物质     

水解蛋白注射液与脂肪乳注射液存在配伍禁忌

水解蛋白在能量供给充足的情况下，参与蛋白质的合成代谢，促进组织愈合，恢复正常生理功能，用于手术严重创伤、大面积烧伤引起的严重氨基酸缺乏，以及各种疾病引起的低蛋白症。脂肪乳为白色乳状液体，用于需要接受胃肠外营养／或脂肪酸缺乏的患者。临床上常将这两者药物配合治疗手术后几日内不能进食的患者，但临床观察发现，水解蛋白与脂肪乳接续静脉输注时，莫菲氏滴管内立刻出现白色液体中悬浮大量小颗粒。为此，将两种药液进行实验，取水解蛋白注射液2ml及脂肪乳注射液2ml吸入同一注射器内，乳白色混合液中即出现大量颗粒状物质，放置24小时颗粒状物质沉淀，未消失。     

备用根大鼠脊髓后角组织神经营养活性物质的分离纯化和生物活性测定

<正> 我室新近的研究发现,部分去传入纤维支配的脊髓后角组织提取液含有神经营养活性组份,其分子量约在40KD～78KD之间,但不清楚是那种分子量的蛋白质具有神经营养活性作用.为了寻找这种物质,本研究应用Superdex G-75prep grad凝胶层析、高效液相色谱(HPLC)层析和细胞培养等技术和方法,进一步分离纯化备用根大鼠部分去传入纤维支配(手术侧)的脊髓后角组织提取液,以期获得某种神经营养活性物质.用SD雄性大鼠100只,切除其一侧L_1-L_3、L_5和L_6背根,仅保留L_4背根(此动物为备用根模型大鼠),术后5天处死动物.分别切取L1_～L_6节段的手术侧和非手术侧脊髓后角组织,制备成提取液、凝胶层析洗脱液和PHLC层拆洗脱液.结果发现,手术侧脊髓后角组织提取液具有神经营养活性作用,将该提取液经Superdex G-75prep grade凝胶层析和PHLC层析后,得到的A峰洗脱液具有促进体外培养鸡胚背根节(DRG)分离神经元存活及其神经突起生长的神经营养活性作用.经SDS-聚丙烯酸胺凝胶电泳(PAGE)显示,A峰洗脱液呈现一条分子量约为60kD的蛋白质主带.结合生物活性检测结果,我们推测分子     

胶质细胞源性神经营养因子与脊髓再生

前　　言胶质细胞源性神经营养因子 (glial cell line- derivedneurotrophic factor,GDNF)是在 1993年由大鼠胶质瘤细胞系 B49中分离的糖基化的二硫键结合的同二聚体蛋白质 ,含有 134个氨基酸 ,分子量为 33～ 35 k D。因其具有 7个保守的半光氨酸残基 ,并且它们在分子出现的位置与转化生长因子- β(transform ing growth factor- β,TGF- β)超家族的全体成员均相同 ,所以 GDNF被认为是 TGF- β超家族的一员。人和大鼠的 GDNF基因业已被克隆 ,二者的氨基酸序列有 93%的同源性 [1 ] 。 GDNF的受体是多成分的复合物 ,它是由固定于质…     

氨基酸分析仪快速测定尿中3—甲基组氨酸含量

3—甲基组氨酸是反映机体蛋白质营养状态的有效指标。本文报告了通过改变121MB型氨基酸分析仪( Beckman公司出品)的控制程序,及对流动相的调整,使单独分     

糖尿病大鼠胰腺差异表达蛋白质组分析

目的 运用蛋白质组学方法,对糖尿病大鼠胰腺蛋白质的表达进行分析。方法用链脲佐菌素（STZ）诱发建立1型糖尿病大鼠模型。用双向凝胶电泳（2D-PAGE）分离大鼠胰腺组织蛋白质,图像分析软件对比分析大鼠的双向电泳图谱,差异表达蛋白经胶内酶切,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)分析获得肽质量指纹图,使用Mascot方法进行检索鉴定。结果 对11个差异表达蛋白质进行分析鉴定,其中4个蛋白质得到有意义结果。结论 糖尿病大鼠胰腺蛋白质组的分析,有助于探索糖尿病的发病机制。     

低聚肽调节免疫功能及其对冠状病毒感染的影响

低聚肽是蛋白质分解为氨基酸过程中的中间产物,其不仅具有蛋白质的营养支撑作用,还具有强大的调节抗氧化和免疫功能的作用。本文就动物、植物和微生物来源的低聚肽对免疫系统的调控作用进行了论述,对其提高免疫系统功能及对冠状病毒感染的防御作用进行了分析,旨在为更合理地利用不同来源和性质的低聚肽制备而成的复合肽类产品,更有效地增强人体免疫功能,为预防和治疗新型冠状病毒肺炎(COVID-19)提供营养支撑。     

阿苯哒唑对鼠体内旋毛虫幼虫蛋白质、氨基酸及元素含量的影响

目的 :探讨阿苯哒唑对旋毛虫幼虫的驱虫机理。方法 :将感染旋毛虫的小鼠分为对照组和服用阿苯哒唑药物的实验组 ,分别对两组鼠肌肉中提取的旋毛虫幼虫进行了蛋白质、氨基酸及所含元素的检测。结果 :实验组和对照组氨基酸含量的变化表明 :用药前后小鼠的 18种氨基酸含量中 ,分别有 5种氨基酸的含量发生了显著性变化 ;实验组和对照组蛋白质含量的变化表明 :实验组蛋白质含量较对照组含量有明显减少 ;通过比较两组元素含量表明 :10种宏量及微量元素中有 8种元素含量有显著性变化。结论 :阿苯哒唑对鼠体内旋毛虫幼虫主要营养和能量来源的的蛋白质、氨基酸及元素含量有明显的影响 ,干扰虫体正常的代谢功能     

Molecular cloning of a gene (poIA) coding for an unusual DNA polymerase I from Treponema pallidum

The gene coding for the DNA polymerase I from Treponema pallidum, Nichols strain, was cloned and sequenced. Depending on which of the two alternative initiation codons was used, the protein was either 997 or 1015 amino acids long and the predicted protein had a molecular mass of either 112 or 114 kDa. Sequence comparisons with other polA genes showed that all three domains expected in the DNA polymerase I class of enzymes were present in the protein (5'-3' exonuclease, 3'-5' exonuclease and polymerase domains). Additionally, there were four unique insertions of 20-30 amino acids each, not seen in other DNA polymerase I enzymes. Two of the inserts were near the boundary of the two exonuclease domains and the other two interrupted the 3'-5' exonuclease domain which is involved in proofreading. The predicted amino-acid sequence had an exceptionally high content of cysteine (2.4% compared with <0.05% for most other sequenced DNA polymerase I enzymes). The polA gene was further cloned into pProEXHTa for expression and purification. The transformants expressed a protein of 115 kDa. Antibodies raised against synthetic peptide fragments of the putative DNA polymerase I recognised the 115-kda band in Western blot analysis. No DNA synthesis activity could be demonstrated on a primed single-stranded template. Although significant quantities of the protein were produced in the host Escherichia coli carrying the plasmid, it was not capable of complementing a polA(-) mutant in the replication of a polA-dependent plasmid.     

一株鹅H5N1亚型流感病毒基因特性的分析

目的 弄清了Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）毒株对鹅致病的分子生物学基础 ，研究香港区人群中发生的禽（Ｈ５Ｎ１）流感的病因，方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，产物纯化，采用双脱链末端终止法测定核苷酸序列，结果 Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）与Ａ／ＨＫ／１５６／９７（Ｈ５Ｎ１）毒株ＲＮＡ４核苷酸序列有２２个位点不同（同源性为９８．８％）无任何掉失或插入。它与人和     

A unique amino-terminal sequence predicted for the chicken proto-ets protein

Avian cellular sequences homologous to the ets domain of the avian leukemia retrovirus, E26, have been characterized, and shown to contain nine discrete regions within a single locus of about 60 kb. The structure of the viral homologous and nonhomologous domains of this chicken ets-1 gene is characterized and seen to define the unique amino acid sequences of the ets protein. We have isolated and sequenced an avian ets-1 cDNA clone obtained from chicken thymus. This cDNA clone contains an open reading frame (ORF) encoding the normal cellular product of 441 amino acids. This product is significantly smaller than that encoded by the v-ets domain of the E26 transforming protein, p135, which contains 491 amino acids. The open reading frame predicted by our sequence data results in a protein calculated to be 50 kDa, which is slightly smaller than that actually observed in chicken cells. The presence of termination codons 5' to this ORF demonstrates that the cDNA characterized contains the entire coding region for the chicken ets gene.     

Full-length sequence analysis of four IBDV strains with different pathogenicities

Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as partial sequences may not designate a particular strain as very virulent, classical, or variant.     

Cloning of a Salmo salar interleukin-1 receptor-like cDNA

The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily, defined by a cytosolic Toll/IL-1R (TIR) signalling domain, participates in host responses to injury and infection. We describe in this study the cloning of a cDNA encoding a Salmo salar interleukin-1 receptor-like protein (SalIL-1RLP). SalIL-1RLP comprises a potential signal peptide, three extracellular immunoglobulin domains, a short transmembrane region and an intracellular region that contains the TIR domain. The predicted amino acid sequence of SalIL-1RLP displays 43-44% similarities and 31% identities to chicken and human IL-1RI sequences. Within the intracellular region, SalIL-1RLP displays highest similarity (59%) and identity (46%) to the chicken IL-1RI sequence. Two different 5' distal UTRs were identified among six salmon IL-1RLP clones. The six clones, however, displayed identical 5' proximal UTRs, coding regions and 3' UTRs. SalIL-1RLP expression is induced in liver, head kidney, spleen and gills upon injection of salmon with bacterial lipopolysaccharide. Sequence comparisons, protein domain structures, expression patterns and phylogenetic analyses indicate that SalIL-1RLP is most closely related to type I interleukin-1 receptors and interleukin-1 receptor related proteins.     

Effect of parenteral nutrition on protein synthesis and liver fat metabolism in man

We studied the effect of parenteral nutrition with amino acids and hypertonic glucose on protein synthesis and liver fat metabolism. Patients with operable gastrointestinal tract malignancies were divided into two groups. Group I ate the hospital diet ad libitum for the 7-10 days preceding surgery. Group II were given adjuvant parenteral nutrition (APN) for 7-10 days prior to the surgical removal of the tumor. Daily nutrient intake and nitrogen balance were determined. [15N[glycine (1-2 g) was infused at a constant rate for 12-18 prior to surgery. During surgery, blood, liver, and muscle specimens were taken for 15N analysis. Fractional protein synthesis rates were estimated by the method of Garlick et al. (Biochem. J. 136: 935-945, 1973). The fat content and distribution pattern in the liver was determined by gas chromatography-mass spectrometry. The following results were found. 1) APN increaed the albumin synthesis rate. 2) The fraction of linoleate in the total liver fatty acids were reduced by 75% in the APN patients. 3) Some of the APN patients developed fatty livers during the study. When the APN patients were subdivided on the basis of whether they had fatty livers, it was found that only those patients who did not accumulate fat showed an improvement in their plasma albumin concentration during the period of parenteral nutrition.     

Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.     

Compensatory renal hypertrophy in hypophysectomized rats

1. After hypophysectomy, both body and kidney weights fall, but at different rates. The rate at which the kidney decreases in weight is faster than that of the whole body.2. Seven days after unilateral nephrectomy, the dry weight of the remaining kidney of hypophysectomized rats, with the exception of rats which had been hypophysectomized for 2 days only, was always heavier than the kidney of control hypophysectomized rats of similar body weight.3. The difference between the dry weight of kidneys of unilaterally nephrectomized hypophysectomized and control hypophysectomized rats increased from 15% in early hypophysectomized (9 days) to about 35% in late hypophysectomized animals (23 days).4. The implantation of renal cortical cells from 2 day hypophysectomized rats into unilaterally nephrectomized control litter-mates inhibited compensatory renal hypertrophy in the latter. When a similar operation was made using kidney cells from animals which had been hypophysectomized for 23 days, there was no significant inhibition of compensatory renal hypertrophy.5. The renal contents of adenosine-3',5'-monophosphate (cyclic AMP) and of guanosine-3',5'-monophosphate (cyclic GMP) in rats hypophysectomized for 2 days were of the same order as those in normal rats, but were markedly lower in rats hypophysectomized for 23 days.6. In contrast to what had been observed in normal rats, in hypophysectomized (2 or 23 days) rats, unilateral nephrectomy did not affect significantly the levels of cyclic nucleotides in the remaining kidney.7. Cross-circulating anephric normal rats with 2 day hypophysectomized animals resulted in an increase of cyclic GMP content in their kidneys. The cross-circulation between anephric normal rats and 23 days hypophysectomized rats had no effect on the level of renal cyclic GMP of the latter.8. When rats hypophysectomized for either 2 or 23 days and which had been nephrectomized were cross-circulated with normal rats, there were no changes in the content of cyclic GMP in the kidneys of the latter.     

HN protein of Newcastle disease virus causes apoptosis in chicken embryo fibroblast cells

Newcastle disease virus (NDV), an avian paramyxovirus, induces apoptosis in chicken embryo fibroblast (CEF) cells. In the present investigation, the ability of haemagglutinin-neuraminidase (HN) protein of NDV to cause apoptosis in CEF cells was examined. The results revealed that cells expressing the HN protein demonstrated decreased DNA content, phosphatidylserine exposure and increased cytoplasmic vacuolation. Up-regulation of caspase-1, -9, -8, -3, loss of mitochondrial transmembrane potential and an increase in oxidative stress were also observed in cells expressing the HN protein. Based on the above results it can be concluded that HN protein of NDV causes apoptosis in CEF cells.