Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

水罐疗法联合白头翁汤灌肠对溃疡性结肠炎患者Th1/Th2免疫平衡及肠黏膜屏障功能的影响

摘要 目的：探讨水罐疗法联合白头翁汤灌肠对溃疡性结肠炎（UC）患者Th1/Th2免疫平衡及肠黏膜屏障功能的影响。方法：选取2020年2月~2022年5月期间湖南中医药大学第一附属医院收治的UC患者100例。根据随机数字表法分为对照组（n=50）和研究组（n=50）。对照组患者接受白头翁汤灌肠,研究组在此基础上接受水罐疗法。对比两组疗效、中医证候评分、Th1/Th2免疫平衡及肠黏膜屏障功能变化情况。结果：研究组的总有效率明显高于对照组（P<0.05）。治疗后,两组里急后重、身热不扬、腹泻黏液脓血便、小便短赤、肛门灼热、腹痛、口干口苦等症状评分均降低,且研究组低于对照组同期（P<0.05）。治疗后,两组Th1/Th2相关指标白介素（IL）-2、γ-干扰素（IFN-γ）均降低,且研究组较对照组低;IL-4、IL-10均升高,且研究组较对照组高（P<0.05）。治疗后,两组肠黏膜屏障功能指标二胺氧化酶（DAO）、D-乳酸（D-LA）均降低,且研究组低于对照组同期（P<0.05）。结论：水罐疗法联合白头翁汤灌肠治疗UC患者,总有效率高,可缓解中医证候,疗效显著,对调节患者的Th1/Th2免疫平衡及肠黏膜屏障功能具有重要作用。     

EXTRACTION,ASSAY AND SOME PROPERTIES OF FLORIDOSIDE PHOSPHATE SYNTHASE FROM PORPHYRA PERFORATA (RHODOPHYTA)1

A suitable method for extraction of floridoside phosphate synthase (FPS, UDP-galactose: sn-3-glycerol phosphate: 1→2′α-D-galactosyl transferase)from Porphyra perforata J. Ag. was developed. Two assay methods for enzyme activity were utilized, one measuring the amount of floridoside formed by using gas-liquid chromatography, the other measuring the sn-3-glycerol phosphate-dependent formation of UDP; both assays gave similar results. FPS is a soluble protein, and FPS activity in the extract as determined by the amount of product formed in vitro compared well with the in vivo rate of floridoside synthesis (4–7 μMmol product formed·h^?1·g^?1 fresh wt). The rate of product formation in vitro was linear up to 45 min and proportional to protein concentration in the assay mixture. The temperature optimum was 30–35° C. FPS was active over a range of pH values from 7.0–8.5. It was stable in concentrated solutions in the presence of 0.3 M ammonium sulfate, but activity was lost in diluted solution (protein concentration below 0.2 mg·mL^?1) or below 0.2 M ion strength. The data suggest that FPS may be an oligomeric protein which occurs free in the cytoplasm or loosely bound to a membrane. It may also be a regulatory protein controlling the overall rate of synthesis of floridoside in vivo.     

REPRODUCTION IN MALE ATLANTIC WALRUSES (ODOBENUS ROSMARUS ROSMARUS) FROM THE NORTH WATER (N BAFFIN BAY)

Age at sexual maturity and timing of the mating season were determined in male Atlantic walruses ( Odobenus rosmarus rosmarus , L.) from the "North Water" subpopulation in northern Baffin Bay. Testes and epididymides of 174 male walruses (between 0 and 30 yr old) from NW Greenland (1987–1990) were studied macroscopically and a subset of 57 specimens was analyzed microscopically. In physically mature bulls ( i.e. , ≥12 yr old), sperm or apparently ripe spermatids were found between 9 November and 12 July. In younger walruses these signs of fertility were found in a few specimens (7–11 yr old) collected between 9 January and 28 May. The mating season seems to peak in January—April. The youngest sexually mature individual was 7 yr old and the oldest apparently immature individual was 13 yr old. Average age of sexual maturity was 10.9 yr (95% C.I.: 9–6–12.2 yr) and all were sexually mature by the time they were 14 yr old. The non-spermiogenetic testes and epididymides showed accelerated growth between about the 5–6th and about the 12–15th year of life, indicating that sexual maturation occurs during these years. The length of the baculum increased gradually until about 12–15 yr of age, when physical maturity was reached.     

Responses in stomatal conductance to elevated CO2 in 12 grassland species that differ in growth form

Responses in stomatal conductance (g _st ) and leaf xylem pressure potential ( _leaf ) to elevated CO₂ (2x ambient) were compared among 12 tallgrass prairie species that differed in growth form and growth rate. Open-top chambers (OTCs, 4.5 m diameter, 4.0 m in height) were used to expose plants to ambient and elevated CO₂ concentrations from April through November in undisturbed tallgrass prairie in NE Kansas (USA). In June and August, _leaf was usually higher in all species at elevated CO₂ and was lowest in adjacent field plots (without OTCs). During June, when water availability was high, elevated CO₂ resulted in decreased g _st in 10 of the 12 species measured. Greatest decreases in g _st (ca. 50%) occurred in growth forms with the highest potential growth rates (C₃ and C₄ grasses, and C₃ ruderals). In contrast, no significant decrease in g _st was measured in the two C₃ shrubs. During a dry period in September, reductions in g _st at elevated CO₂ were measured in only two species (a C₃ ruderal and a C₄ grass) whereas increased g _st at elevated CO₂ was measured in the shrubs and a C₃ forb. These increases in g _st were attributed to enhanced _leaf in the elevated CO₂ plants resulting from increased soil water availability and/or greater root biomass. During a wet period in September, only reductions in g _st were measured in response to elevated CO₂. Thus, there was significant interspecific variability in stomatal responses to CO₂ that may be related to growth form or growth rate and plant water relations. The effect of growth in the OTCs, relative to field plants, was usually positive for g _st and was greatest (>30%) when water availability was low, but only 6–12% when _leaf was high.The results of this study confirm the importance of considering interactions between indirect effects of high CO₂ of plant water relations and direct effects of elevated CO₂ on g _st , particularly in ecosystems such as grasslands where water availability often limits productivity. A product of this interaction is that the potential exists for either positive or negative responses in g _st to be measured at elevated levels of CO₂.     

Stereospecificγ-lactamase activity in aPseudomonas fluorescens species

Twenty five environmental isolates enriched for their ability to grow onN-acetylphenylalanine as sole carbon source were investigated for their hydrolytic action on (+)-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one). Strain CMC 3060, a mucoidal Gram-negative rod identified as a strain ofPseudomonas fluorescens, produced high levels of (+)lactamase, and was subsequently found to produce two distinct intracellular enantiomer-selective, -lactamases, one for each isomer. The (+)lactamase was produced constitutively whereas the (-lactamase was produced only in the presence of the substrate. The (+)lactamase was stable when stored as a frozen cell paste but unstable as a protein solution, losing activity during purification and storage. This enzyme was highly selective for the (+)lactam and showed no activity against a wide range of similar compounds. By use of rapid purification techniques and the inclusion of protease inhibitors and protein stabilisers, the (+)lactamase was purified to homogeneity by FPLC and found to be a monomer of molecular weight 61000 Da.     

单抗在人铁蛋白免疫亲和纯化中的应用

抗人铁蛋白单抗６Ｄ６和Ａ－ｈＦ－Ｃ与肝型铁蛋白和心型铁蛋白的反应性有所不同。６Ｄ６对两种铁蛋白的反应性相似;而Ａ－ｈＦ－Ｃ单抗与肝型铁蛋白的反应性较强。我们将６Ｄ６和Ａ－ｈＦ－Ｃ分别制成了亲和凝胶,用来纯化人肝脏和心脏粗抽提物中的铁蛋白。此法具有操作简便,产率和产品纯度高等优点。     

Water molecule binding and lifetimes on the DNA duplex d(CGCGAATTCGCG)2

Summary Measurements of the water proton spin-lattice relaxation rate for aqueous solutions of the palindromic dodecamer, d(CGCGAATTCGCG)₂, are reported as a function of the magnetic field strength. The magnitude of the relaxation rates at low magnetic field strengths and the shape of the relaxation dispersion curve permit assessment of the number of water molecules which may be considered bound to the DNA for a time equal to or longer than the rotational correlation time of the duplex. The data are examined using limiting models that arbitrarily use the measured rotational correlation time of the polynucleotide complex as a reference point for the water molecule lifetime. If it is assumed that water molecules are bound at DNA sites for times as long as or longer than the rotational correlation time of the duplex, then the magnitude of the relaxation rates at low field require that there may be only two or three such water sites. However, if the lifetime constraints is relaxed, and we assume that the number of water molecules bound to the DNA is more nearly the number identified in the X-ray structures, then the average water molecule lifetime is on the order of 1 ns. Measurements of ¹H NOESY spectra demonstrate that some water molecules must have lifetimes sufficiently long that negative Overhauser effects are observed. Taken together, these results suggest a distribution of water molecule lifetimes in which most of the DNA-bound water molecule lifetimes are shorter than the rotational correlation time of the duplex, but where some have lifetimes of at least 1 ns under these concentrated conditions.Abbreviations DNA deoxyribonucleic acid - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy     

二氢叶酸还原酶结合底物的去除

分析了应用氨甲蝶呤（MTX-Agarose）亲和层析法提纯的鸡肝二氢叶酸还原酶的组成和性质.建立了用平面粒度胶等电聚焦法去除与酶紧密结合底物的方法．讨论了结合底物对酶构象研究的影响,并指出,用未完全去除结合底物的酶研究酶在变性过程构象变化会得到错误的结论．     

北方稻田生态系统水量平衡及水分效率研究

１９９３～１９９５年研究了５种不同模式水稻田生态系统水量平衡及水分效率结果表明,不同水稻田模式其总耗水量之间有明显差异,其中节水模式和节水节肥模式较常规模式节省灌溉水达１５～２３％,水分生产效率增加３０％以上．各模式蒸发蒸腾耗水量在同一生长季内基本相同．田间结构及调控管理对其无明显影响实测水稻生育期田间蒸发蒸腾量与计算的可能蒸发蒸腾量相差不过５％。