正常人及肝细胞癌患者肝脏及血浆中丙酮酸激酶同工酶免疫测定

 分别从人肝及鼠肝中高度纯化L型和M_1型丙酮酸激酶(Pyk),制备相应的免抗血清。从抗血清中纯化IgG并水解成F(ab')_2,进而还原成Fab',后者与辣根过氧物酶(HRP)交联成Fab'-HRP,再利用这两种复合物建立了各自的高灵敏夹心ELISA来免疫定量L及M_2型(与鼠M_1-Pyk有交叉免疫)Pyk,结果发现:正常人肝中95％的Pyk为L型,M_2-Pyk仅占5％,而肝细胞癌中L-Pyk降至正常肝的3.5％,M_2-Pyk却增至正常的14.6倍,以致M_2-Pyk占总量的95.5％。免疫组化研究进一步证实定量的结果,正常人肝L-Pyk染色呈强阳性。M_2-Pyk染色较弱,而肝细胞癌恰好相反,L-Pyk染色明显减退,而M_2-Pyk不仅癌组织染色很深,癌周组织也明显深于正常,而且愈近癌巢,染色愈深。测定血浆Pyk,发现正常人L-Pyk占89.9％,M-Pyk(以M_2计)仅占10.1％。肝细胞癌患者血浆L-Pyk略见降低,为正常值的79.6％,p值在0.02～0.05之间,但血浆M型Pyk明显升高至正常的4.59倍,占Pyk总量的39.6％,并证明主要来源于肝癌组织中的M_2,故M_2-Pyk有可能成为肝细胞癌诊断的新指标。     

Prefabricated buccal mucosa-lined flap in an animal model that could be used for vaginal reconstruction

Congenital vaginal aplasia, gynecological tumor excision, and male-to-female sex surgery are three clinical conditions in which the plastic surgeon is involved in vaginal reconstruction. Skin-lined or skin-grafted local flaps are currently used, but for many reasons, keratinized skin is not the ideal lining for such a moist cavity because it leads to dryness, desiccation, maceration of the skin, and even hair growth in the cavity. The purpose of this study was to create a subcutaneous cavity lined with mucosa in an area with a predictable blood supply. The abdominal area supplied by the deep circumflex iliac vessels was chosen. Six minipigs were used. Strips of tongue buccal mucosa formed the lining; if additional tissue was required, it was taken from the mucosal aspect of the cheek. The mucosa was expanded by using multiple stab incisions. The mucosa was sutured onto the fascia supplied by the deep circumflex iliac vessels, and the skin incision was closed over a silicone sheet to prevent adhesion to the underlying mucosa. This was left for 1 week to allow the mucosa to take. The prefabricated fascial flap was rolled over a silicone stent and was closed longitudinally to form a cylindrical shape. The flap was placed in a subcutaneous pocket in the right inguinal area. The caudal end was left open and was sutured to the surrounding skin. The silicone stent was used to keep the cavity patent and to prevent adhesions in the early stage of the healing process. Regular digital examination was performed to assess patency and contour; endoscopy allowed assessment of mucosa viability. This method of producing a mucosa-lined flap may provide a solution to the difficult problem of vaginal reconstruction.     

Flower-inducing and -inhibiting activities of Phloem Exudate from Cotyledons of Pharbitis Seedlings

The flower-inducing and -inhibiting activities of phloem exudate (PE) prepared from cotyledons of Pharbitis seedlings were examined, using apex cultures in vitro from Pharbitis as a bioassay system.The PE was prepared from photoperiodically-induced cotyledons (SD-PE). The SD-PE was subjected to the following fractionations: When the SD-PE was extracted with CHCl₃ and then ethyl acetate, the inducing activity was located in the final aqueous fraction. The activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). The diffusate was fractionated by ion exchange chromatography, and flower-inducing activity was found in the fraction adsorbed onto anion exchange resin. When the fraction was applied to a Sep-Pak C18 cartridge, the activity eluted with 25% MeOH. As a result of the above fractionation, activity was increased about 30-fold.The nature of the flower-inhibiting activity of the PE taken from cotyledons exposed to continuous-light conditions was examined (CL-PE). The inhibiting activity was decreased as the cotyledons were exposed to longer dark periods; it appeared to be heat-stable. The CL-PE also inhibited flowering in Lemna. The CL-PE was subjected to the following fractionations: When the CL-PE was extracted with CHCl₃ and ethyl acetate, activity was located in the final aqueous fraction. Activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). When the diffusate was fractionated by ion exchange chromatography, the activity was found in the flow-through fraction. When the fraction was applied to a hydroxyapatite cartridge, the activity eluted with 25 mM sodium phosphate buffer. When the fraction was re-dialyzed (molecular weight cut off was 1,000), the diffusate contained the activity. As a result of the above fractionation, activity was increased about 10-fold.     

Purification and some properties of beta-N-acetyl-D-glucosaminidase from the cabbage butterfly (Pieris rapae)

A beta-N-acetyl-D-glucosaminidase (NAGase) from the cabbage butterfly (Pieris rapae) was purified. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was determined to be 106 kDa by gel filtration, and the result of SDS-PAGE showed that the enzyme was a heterodimer, which contained two subunits with different mass of 59.5 and 57.2 kDa. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 degrees C, respectively, and the Michaelis-Menten constant (K(m)) was determined to be 0.285 mM at pH 6.2 and 37 degrees C. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 degrees C. The activation energy was 83.86 kJ/mol. The reaction of this enzyme with pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products. The ionization constant, pK(e), of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 degrees C, and the standard dissociation enthalpy (DeltaH(o)) was determined to be 2.18 kcal/mol. These results showed that the ionizing group of the enzyme active center was the carboxyl group. The results of chemical modification also suggested that carboxyl group was essential to the enzyme activity. Moreover, Zn(2+), Hg(2+), Cu(2+) had strongly inhibitory effects on the enzyme activity.     

Birth of a foal after oocyte transfer to a nonovulating, hormone-treated recipient mare

A nonovulating, hormone-treated mare was used successfully as an oocyte recipient. The mare's ovarian activity was suppressed using progesterone and estrogen treatment. This treatment was stopped, then estrogen was administered for 3 d prior to the transfer. An oocyte was recovered from the follicle of a donor mare and was transferred via flank laparotomy into the recipient's oviduct. The recipient mare was inseminated 7 h before transfer. The recipient was treated with intramuscular progesterone from the day after transfer until 47 d after transfer, and then with oral altrenogest until 150 d gestation. A normal colt was born at 321 d gestation, and was shown by DNA analysis to be the progeny of the donor mare. This is the first report of fertilization and embryo development to term after transfer of oocytes to a nonovulating mare, and, to our knowledge, the first of its kind in any domestic species.     

Purification and properties of haloalkane dehalogenase from Corynebacterium sp. strain m15-3

A haloalkane dehalogenase was purified to electrophoretic homogeneity from cell extracts of a 1-chlorobutane-utilizing strain, m15-3, which was identified as a Corynebacterium sp. The enzyme hydrolyzed C2 to C12 mono- and dihalogenated alkanes, some haloalcohols, and haloacids. The Km value of the enzyme for 1-chlorobutane was 0.18 mM. Its molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 33,000 by gel filtration. The isoelectric point was pH 4.5. The optimum pH for enzyme activity was found to be 9.4, and the optimum temperature was 30 to 35 degrees C. The enzyme was stable for 1 h at temperatures ranging from 4 to 30 degrees C but was progressively less stable at 40 and 50 degrees C.     

高校《普通动物学》教学中实施自主学习的探索

课堂教学是学生获取知识的主渠道,教会学生如何自主学习已经成为世界各国基础教育的根本任务之一。在现代社会自主学习是学习者所必备的重要能力之一,在课堂教学模式下,让学生走自主学习之路是学会学习的有效途径。作者对本校《普通动物学》课堂教学模式下实施自主学习现状进行了分析、对比、改进,目的在于培养学生自主学习的意识,增强自主学习能力,提高学习效率。结果证明学生在自主式教学模式下积极性被充分调动,学习成绩显著提高。     

Ammonia formation in isolated rat liver mitochondria

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutamate transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix glutamate except when the matrix pH was unphysiologically low. The data suggest that the Km of matrix glutamate for efflux is decreased by H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the Km of medium glutamates and Vmax. Mitochondria were incubated at 15 and 28 degrees C with glutamate and malonate. Under these conditions, glutamate was metabolized only by the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix alpha-ketoglutarate was varied either by the omission of malonate from the incubation media or by adding alpha-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evaluate the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. alpha-Ketoglutarate was found to be a potent competitive inhibitor of ammonia production and glutamate dehydrogenase. Inhibition of glutamate dehydrogenase by alpha-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.     

Chemical forms of selenium in selenium containing proteins from human plasma

The chemical forms of selenium (Se) were determined in human plasma fractions. Human plasma was subjected to gel filtration using Sephadex G-150, and the first Se peak from this column was subsequently chromatographed on DEAE-Sephacel. The form of Se in the Se peak which eluted from this column was shown to be selenocysteine (SeCys). In a second approach human plasma was again subjected to gel filtration and the first Se peak was chromatographed on Affigel blue. SeCys was shown to be the form of Se in both the retained and unretained Se on this column. The second gel filtration Se peak was also chromatographed on Reactive Blue 2-Sepharose CL-6B and the form of Se which was not retained was also shown to be SeCys. However, the form which was retained was shown to be selenomethionine. Evidence is presented that there are three Se containing proteins in human plasma, which are selenoprotein P, glutathione peroxidase, and albumin.     

超声波法提取剑花总皂苷工艺研究

利用超声波法,采用正交实验对剑花总皂苷提取工艺进行研究。结果表明,提取剑花总皂苷的最优条件为:粒度80目,最佳溶剂水,超声时间40 min,溶剂挥干温度80℃,料液比1:20。     

上呼吸道感染患儿咽部病原菌分布及耐药性分析

目的了解天津滨海地区儿童上呼吸道感染病原菌情况及耐药性,以便指导临床合理用药。方法对2012年1月至2013年4月天津市滨海新区汉沽中医医院住院儿科1022例上呼吸道感染儿童咽拭子标本进行培养及鉴定。采用法国ATB自动细菌鉴定仪及MIC药敏板进行细菌鉴定和药敏试验,β-内酰胺酶试验采用头孢硝噻吩纸片法,耐甲氧西林金黄色葡萄球菌（MRSA）采用头孢西丁纸片法检测。结果检出病原菌296株,其中金黄色葡萄球菌、肺炎链球菌及流感嗜血杆菌检出率分别为13. 70%、5. 97% 、5. 19%,分列前三位。金黄色葡萄球菌对常用抗菌药物耐药率为苯唑西林30. 0%、青霉素95. 7%、红霉素80. 0%、左氧氟沙星4. 3%,MRSA占30. 0% （42/140）、;肺炎链球菌对常用抗菌药物耐药率为青霉素9. 84%、红霉素54. 1%、克林霉素52.5%、左氧氟沙星3. 3% ;流感嗜血杆菌对常用抗菌药物耐药率为氨苄西林34. 0%、阿奇霉素0%、左氧氟沙星0%,复方新诺明56. 6%、头孢呋辛3. 8%、阿莫西林/克拉维酸5. 7%。结论天津滨海地区儿童上呼吸道感染病原菌分布广泛,检出致病菌有一定的耐药率,临床应重视根据病原菌感染情况及药敏试验结果,进行耐药性监测和合理用药。     

Nutrient-split feeding strategy for dialysis cultivation of Escherichia coli

Reduction in nutrient loss during dialysis cultivation of Escherichia coli on a glycerol medium was investigated. A dialysis reactor with an inner fermentation and an outer dialysis chamber was used. Aerobic condition was maintained by limiting the glycerol feed rate to an optimum value which was estimated from the oxygen requirements for glycerol oxidation and oxygen transfer capacity of the reactor. High reduction in nutrient loss was achieved by using water as the dialyzing fluid. However, osmotic movement of water from the dialysis to the fermentation chamber was observed, and the final cell concentration was low. With a nutrient-split feeding strategy (feeding glycerol directly to the fermentation chamber and dialyzing with salt solution), glycerol loss was small, there was no osmotic flux of water to the fermentation chamber, and the cell concentration was high. Both glycerol and salt loss could be avoided, and a cell concentration of 170 g/L was obtained when the dialysis process was substituted by addition of XAD adsorbents to the dialysis chamber. Application of this nutrient-split feeding strategy to cell cultivation in a stirred tank reactor, coupled with dialysis in external dialyzer modules, resulted in low cell concentrations. (c) 1993 Wiley & Sons, Inc.     

Osmoregulation in the parasitic protozoan Tritrichomonas foetus

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K(+) concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K(+) concentrations and was inhibited when K(+) and/or Cl(-) was absent from the medium. The results suggested that the well-documented Na(+)-K(+)-2Cl(-) cotransport system was responsible for the K(+) influx activated during the RVI. However, inhibitors of Na(+)-K(+)-2Cl(-) cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K(+) influx in T. foetus.     

Cellulase production by Trichoderma reesei when cultured on xylose-based media supplemented with sorbose

The ability of L-sorbose to stimulate cellulase production In shake flask culture of Trichoderma reesei was examined in mineral salts media (initial pH 5.0) containing either 1.0% D-xylose, 1.0% cellulose, and/or 0.1, 0.3, or 0.5% L-sorbose. When sorbose was the only carbon source, growth was limited, little substrate was utilized, pH increased, and cellulase activity was not apparent. The other carbon sources promoted good growth, pH dropped sharply to 2.5-3.0, substrate was utilized rapidly, and cellulase activity was detected. After three weeks of fermentation, twice as much cellulase activity was detected in the medium containing only cellulose as the carbon source, as compared to xylose as the carbon source. Cellulase activity was higher when media contained xylose supplemented with sorbose compared to xylose as the only carbon source. At 0.3 and 0.5% levels of sorbose supplementation of xylose-based media, cellulase activity was similar to that in cellulose-based media.     

Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectrometric analysis after in gel chymotryptic digestion

To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis.     

接种量对花绒寄甲繁殖的影响

释放花绒寄甲防治天牛已成为重要手段,而繁殖花绒寄甲时,接种花绒寄甲幼虫到大麦虫蛹体时,控制合适的接种量是提高花绒寄甲繁殖数量和质量的关键技术。本研究通过在替代寄主上人工接种不同数量的花绒寄甲幼虫,观察其发育情况及其子代数量、质量等指标,明确最佳接种量。结果表明：随着接种量的增加,花绒寄甲幼虫历期和蛹历期明显缩短,其中接种4头/个和6头/个,幼虫历期为13 d,蛹历期为33 d,而接种14头/个和16头/个时,幼虫历期短于12 d,蛹历期明显缩短为28~29 d。花绒寄甲结茧数随接种量增加而增加,接种数为16头/个时,结茧数最多,接近7个。花绒寄甲结茧率随着接种量增多而降低,4头/个时,结茧率最高为72.3%。接种量对花绒寄甲子代个体数量和大小均有显著影响,其中接种8头/个时,羽化数平均为4.3头,显著高于接种4头/个（羽化数平均为2.8头）,明显低于接种16头/个(羽化数平均为6.9头)。接种量为4头/个和6头/个时,子代成虫个体最大,单头重平均每头可达0.035 g,接种量为8头/个时,成虫单头重平均每头0.032 g左右,接种量达到16头/个时,单头重最轻,为0.023 g。对花绒寄甲羽化率无显著影响,7个处理下子代羽化率均较高,平均在94.4%~100%。接种量越少,更利于花绒寄甲的生长发育,当接种量为4头/个时,花绒寄甲成虫发育最好,其子代个体最大,但子代数较少。因此,利用大麦虫蛹繁育花绒寄甲种虫时,最佳接种量为4头/个,而需要规模化繁育花绒寄甲作为天敌使用时,综合考虑子代数量和质量以及经济成本,最佳接种量为8头/个。     

A microbial biosensor for trimethylamine using Pseudomonas aminovorans cells

A biosensor system based on the difference in the oxygen uptake response of two microbial electrodes was developed to monitor trimethylamine (TMA). The first electrode, constructed using Pseudomonas aminovorans grown on TMA, was sensitive to TMA, trimethylamine N-oxide (TMAO), dimethylamine (DMA) and monomethylamine (MMA). The second electrode responding to TMAO, DMA and MMA was prepared using Ps. aminovorans grown on TMAO. The difference in oxygen uptake was linearly related to the TMA concentration in the range of 5-26 microM. The minimum detectable level was 2.6 microM and the relative standard deviation was determined to be 14% for 16 repeated analyses. When operated and stored at 30 degrees C, the response of the system was stable for only 2 days. However, when the biosensor system was operated at 30 degrees C but stored overnight at 4 degrees C, the system was stable up to 20 days. The biosensor system was applicable for the determination of TMA in fish tissue extracts and the results compared well with those determined by HPLC.     

Inhalation anesthesia in adult cattle

An inhalation technique was used for anesthesia during ileal cannulation in five adult cows. Following sedation with intravenous acepromazine, anesthesia was induced intravenously with thiopental sodium in 5% glyceryl guaiacolate solution. Endotracheal intubation was performed and anesthesia maintained with halothane in oxygen via a circle system with a precision vaporizer. In all cases, induction was smooth and no difficulties were experienced during the maintenance of anesthesia. Total anesthesia time was 1.5 to 2.5 hours. Following completion of the surgical procedure, which was performed with the animal in left lateral recumbency, each cow was rolled to a sternal position and supported, if necessary. The endotracheal tube was left in place, with oxygen administration continued, until the animal was able to swallow. Recoveries were rapid and all animals were ambulatory within 30 minutes after completion of the surgery. The only post-operative complication due to anesthesia was transient mouth soreness in two cases, attributed to the use of a mouth speculum during intubation.     

逆相蒸发法制备茶多酚脂质体及质量评价

采用逆相蒸发法制备茶多酚脂质体并进行质量评价。通过二次回归旋转组合设计优化茶多酚脂质体制备工艺及配方,对其形态、结构、粒径分布等性质进行考察。研究结果表明,最佳配方为m（大豆卵磷脂）：m（胆固醇）=3：1、茶多酚质量浓度为7mg／mL、V（有机相）：V（水相）=4：1、磷酸盐缓冲液浓度15mmoL／L,此条件下包封率为50．37％;所制备的茶多酚脂质体形态呈圆球形或椭球形,为大单室脂质体,有效粒径为165．3nm,Zeta电位为-69．3mV。逆相蒸发法制备茶多酚脂质体方法简单可行,所制备的脂质体具有一定缓释性。     

The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.