Immunologically active nonapeptide fragment of a proline-rich polypeptide from ovine colostrum: Amino acid sequence and immunoregulatory properties

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. To study the relationship between the structure of PRP and its immunomodulatory properties, the polypeptide was digested by chymotrypsin. Products of the proteolysis were separated by gel filtration and three fractions were obtained: PRP-1, PRP-2 and PRP-3. The activity of the fractions was compared with the activity of the untreated PRP. It was found that PRP-1 was inactive, whereas PRP-2 and PRP-3 showed an activity in the regulation of the immune response assayed by measurement of PFC, and by studying effects on delayed hypersensitivity, formation of autologous rosette-forming cell, and sensitivity of thymocytes to hydrocortisone. The activity of PRP-2 and PRP-3 was comparable to the activity of PRP. The PRP-3 fraction of low mol. wt was further purified and a pure nonapeptide of mol. wt 1000 (PRP-3b) was isolated. The amino acid sequence of PRP-3b was: Val—Glu—Ser—Tyr—Val—Pro—Leu—Phe—Pro. The nonapeptide showed the full spectrum of biological activities of PRP. Comparison of terminal amino acid suggested that PRP-3b was neither the NH₂- nor the COOH-terminal fragment of PRP. The amino acid sequence of the nonapeptide indicated that PRP-3b is different from other known immunomodulators.     

Antibodies to the first constant-region domain of the murine mu-chain induced by a synthetic peptide

A peptide corresponding to the N-terminal 13 amino acid residues of the murine C mu 1 domain was synthesized by the solid-phase method and was coupled to carrier proteins through an additional cysteine residue. Rabbit antisera to these peptide-carrier conjugates were found to react with intact mouse IgM as well as its Fab mu fragment. These antisera also reacted with the isolated mu-chain and the V mu fragment of the heavy chain. This fragment consists of the VH-domain and the N-terminal residues of the C mu 1 domain preceding the interchain half-cystine. No significant reactivity of these antisera was found with the IgM of human and equine species or with murine IgG isotypes. Apart from their utility in the purification of the V mu fragment, these and similar antisera can be used to probe structure and function relationships of immunoglobulin domains. Furthermore, such antisera may be used in the study of expression vectors with heavy-chain genes to detect the expression of truncated forms of heavy chain in E. coli and other hosts.     

SYNTHESIS,CHARACTERIZATION AND FLUORESCENCE STUDIES ON N-α-DANSYLALANYLLYSYL TRYPSINO (HIS-46)-METHANE

The 1-dimethylamino-5-naphthalene sulfonyl (dansyl) derivative of alanyllsyl chloromethane was synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active site of trypsin via affinity labelling. The potential of dansylalanyllysyl chloromethane lies in its high degree of selectivity and markedly faster rate of enzyme inactivation when compared to previously synthesized, single residue affinity label chromophores. This permits the practical utilization of stoichiometric amounts of the inhibitor to achieve 100% inactivation of trypsin, even at high dilutions. The transfer of energy between the four tryptophan residues of trypsin and the bound dansyl group has been investigated in the fluorescent inhibitor-enzyme conjugate. From transfer efficiency measurements mean distances of 19.0 Å and 19.3 Å between the point of attachment of the dansyl group and the four tryptophan residues of trypsin have been calculated. These compare well with the mean value of 18.8 Å derived from calculations based on crystallo-graphic model studies.     

Development of fluorescent-labeled trapping reagents based on cysteine to detect soft and hard electrophilic reactive metabolites

Trapping assays are conducted at lead optimization stages to detect reactive metabolites (RMs) that can contribute to drug toxicity. The commonly used dansyl glutathione (dGSH) provides a sensitive analysis owing to the fluorescent label, however, it captures only soft electrophilic RMs. TRs for hard electrophilic RMs, few of which are labeled fluorescently, can detect hard electrophilic aldehydes only by forming unstable imine derivatives. In this study, we aimed to develop novel fluorescently labeled TRs that detect both soft and hard electrophilic RMs and form stable ring structures with aldehydes. We designed four dansylated TRs based on cysteine, which has both soft and hard nucleophilic groups. To evaluate the reactivity of the TRs, we incubated them with several substrates and found that one of the TRs (CysGlu-Dan) detected all the soft and hard electrophilic RMs. We also examined the inhibition potential of each TR for seven major CYPs involved in drug metabolism and found that CysGlu-Dan showed an inhibitory profile similar to that of dGSH. In conclusion, CysGlu-Dan can be used to evaluate the risk of RMs in drug discovery.     

人肾心钠素降解酶的分布及心钠素降解抑制的研究

将人肾分为外皮质（ＯＣ）、内皮质（ＩＣ）、外带（ＯＳ）、内带（ＩＳ）、和内髓质（ＩＭ）五个区带，运用荧光法和放免法，对中性肽链内切酶（ＮＥＰ）高度特异的合成代用底物［丹酰－丙氨酰－甘氨酰－对硝基苯丙氨酰－甘氨酸（ＤＡＧＮＰＧ）］和天然底物心钠素研究心钠素降解酶在各区带的分布及心钠素降解的抑制。结果表明：人肾心钠素降解酶的分布，在各区带有所不同，以ＯＣ最高，ＩＭ最低，降解酶活性依次为ＯＣ＞ＩＣ＞ＯＳ＞ＩＳ＞ＩＭ，提示该酶对心钠素的降解主要发生在肾小球和近端小管。抑制实验表明，黄豆胰蛋白酶抑制剂和水稻丝氨酸蛋白酶抑制剂均能有效抑制人肾对ＤＡＧＮＰＧ和心钠素的降解，黄豆胰蛋白酶抑制剂的抑制率分别为７５．７％和７２．０％，水稻丝氨酸蛋白酶抑制剂的抑制率分别为７５．３％和７４．７％。     

柱前衍生法测定胸腺中组胺的含量

目的建立一种科学、合理的检测胸腺中组胺含量的方法,用于控制胸腺原料的质量。方法采用丹磺酰氯柱前衍生,HPLC-UV法,C18（4．6mm×250mm,5μm）色谱柱,乙腈-水为流动相,流速1．0mL·min-1,检测波长254nm,梯度洗脱。结果采用本方法测定组胺标准品,在1．0～50μg·mL-1的范围内,组胺的浓度与峰面积成线性相关,相关系数为0．9979,加样回收率85％～110％,RSD％〈10％。结论采用该方法测定胸腺中痕量组胺的含量,具有简单、快速、准确、灵敏,重复性好的特点,可以用于胸腺原料的质量控制。     

Dansyl chloride labelling of stratum corneum: its rapid extraction from skin can predict skin irritation due to surfactants and cleansing products

The irritation potential of surfactants and body cleansing products was determined by evaluating the removal of dansyl chloride from the skin. Dilute solutions (2% active ingredient, w/v) of surfactants and soap extract fluorescence from the skin within 30 min. This is probably a physicochemical effect as it is too rapid to be due to a modification of epidermal cell turnover rate. Such an extraction of the fluorescent dye occurs without any clinical sign of irritation. However, it may represent an early phase of skin irritation process, because it is related to the ranking of irritant products as determined by other assessment methods.     

高效液相色谱法测定心肌肽中组胺含量的可行性分析

目的 建立和验证高效液相色谱(HPLC)测定心肌肽中组胺含量的方法.方法 采用多道生理记录仪记录受试样品致猫血压下降情况.用丹磺酰氯柱前衍生化高效液相色谱法测定心肌肽中组胺含量,心肌肽溶液经三氯乙酸沉淀后,取上清液以丹磺酰氯丙酮溶液为衍生化试剂,衍生化后进行高效液相色谱测定.色谱条件为:色谱柱C18柱(250mm×4.6 mm,i.d.,5μm),以水-乙腈为流动相梯度洗脱,检测波长254 nm,并对此方法进行方法学考察(标准曲线的绘制、加标、回收率、精密度重复性、专属性).分析样品中组胺含量与致猫血压下降值之间的相关性.结果 l～5号样品dT/dS值分别为(0.22±0.07,0.74±0.07,0.91±0.03,0.93±0.10,1.16±0.05)均为合格产品.组胺对照品在0.1～ 10 μg/mL范围内线性关系良好,相关系数(r) ＞0.996,检出限为0.03 μg/mL,平均加标回收率＞91.1％.日内与日间精密度RSD均＜1.0％,重复性及专属性均符合生物样品分析要求.SPSS软件分析心肌肽中组胺的含量与降压物质检查法中降压反应比值(dT/dS)成良好的线性相关(r=0.974,P=0.036).结论 采用HPLC法测定心肌肽溶液中组胺的含量,并结合降压反应比值(dT/dS)设定组胺含量的限值可能成为控制心肌肽原料药质量的良好方法.     

盐酸美金刚胶囊的丹磺酰氯柱前衍生化-HPLC法测定

建立了柱前衍生化-HPLC法测定盐酸美金刚胶囊的含量.样品与丹磺酰氯在25℃、避光衍生化反应90 min后测定.采用C_8 色谱柱,以磷酸盐缓冲液(pH 3.0)-乙腈(25:75)为流动相,检测波长218 nm.在5～180 μg/ml浓度范围内线性关系良好(r=0.999 9),日内和日间RSD为1.15%和0.48%,平均回收率为99.7%～101.1%,衍生化产物在17h内稳定.