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.     

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。     

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.     

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.     

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.     

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.     

一种新的丙型肝炎病毒单链丝氨酸蛋白酶基因的构建、表达与鉴定

根据丙型肝炎病毒 (HCV)丝氨酸蛋白酶晶体结构特点 ,设计并构建了一种新的单链型丝氨酸蛋白酶分子 .该分子由辅因子NS4A的核心序列、柔性连接子GSGS和NS3丝氨酸蛋白酶结构域组成 .利用设计的 3条引物 ,通过 2轮PCR获得单链丝氨酸蛋白酶基因 ,插入原核表达载体pQE30中 ,转化大肠杆菌M15 ,获得重组克隆 .经低剂量诱导和低温培养 ,目的基因获得高水平可溶表达 .以金属螯合层析法纯化的重组蛋白纯度达 95 %以上 .间接ELISA法检测 98份血清证实 ,该蛋白具有良好的抗原性和特异性 ;以重组蛋白底物NS5ab和单链丝氨酸蛋白酶建立了简便、实用的丝氨酸蛋白酶体外活性检测系统 ;以该系统观察了PMSF和EDTA对蛋白酶活性的影响 .结果表明 ,PMSF能够抑制蛋白酶的酶切活性 ,而EDTA不能抑制酶的活性 .单链型HCV丝氨酸蛋白酶的成功表达以及体外活性检测系统的建立 ,为丝氨酸蛋白酶抑制剂的研制奠定了物质基础 .     

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。     

Comparative toxic and sublethal effects of fluvalinate on two-spotted spider mite and European red mite

Four fluvalinate formulations differed in their residual toxicity to female two-spotted spider mite (TSM), Tetranychus urticae adults; the emulsifiable concentrate (EC) was the most toxic. In contrast, there was little difference in toxicity between the formulations with the European red mite (ERM) Panonychus ulmi with the exception of the EC formulation which was the least toxic. Fluvalinate 2F caused minimal (<10%) TSM and ERM egg mortality. Fluvalinate 2F was more toxic and caused greater larval dispersal for the TSM compared to the ERM at the field concentration and below. The toxicity of fluvalinate 2F to TSM and ERM protonymphs, deutonymphs and adults was low, approximately <20% at field concentration. Dispersal was the main response to fluvalinate and this was positively correlated with increasing concentration. The combined mortality and dispersal LC₅₀ was five times lower for ERM protonymphs and adults, but 11 times higher for ERM deutonymphs compared to equivalent TSM life stages. Fluvalinate 2F reduced TSM development from the protonymph and deutonymph stages to a greater extent compared to the ERM. The mortality response to fluvalinate 2F was unaffected by host type (peach or apple) for the TSM whereas ERM mortality was higher on apple compared to peach. TSM dispersal was higher from apple compared to peach whereas ERM dispersal was similar on both host types. Oviposition by both mite species was lower on apple than peach leaves. A 1 h exposure to fluvalinate 2F reduced ERM oviposition for 12 days.     

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.     

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。     

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.     

一株粘质沙雷氏菌烈性噬菌体污水分离及特性

[目的]以粘质沙雷氏菌(8039)为宿主菌从医院污水中分离噬菌体并对其基本生物学特点进行研究.[方法]四步法污水分离噬菌体;单、双层平板噬菌斑实验筛选烈性噬菌体并观察噬菌斑形态;纯化后2%磷钨酸染色电镜观察;手工法提取噬菌体核酸酶切后琼脂糖凝胶电泳分析;利用双层平板噬菌斑实验测定最佳感染复数和完成一步生长实验.[结果]从医院污水中成功分离出粘质沙雷氏菌烈性噬菌体一株(SM701),该噬菌体有一个正多面体立体对称的头部,头径约64nm,无囊膜,有一长尾,无收缩尾鞘,尾长约143nm;基因组核酸能被双链DNA内切酶BamH Ⅰ及Hind Ⅲ切开,大小约57kb;噬菌斑圆形透明,直径1mm左右(培养12h,),边界清楚;当感染复数(multiplicity of infection,MOI)为10时,子代噬菌体滴度较高;按照一步生长实验结果绘制出一步生长曲线,可知感染宿主菌的潜伏期是约为30min,爆发期约100min,平均爆发量约为630[结论]按照国际病毒分类委员会分类标准,该噬菌体属于长尾噬菌体科(siphoviridae)烈性噬菌体,按照Bradley和Ackermann形态分类法属于B1亚群;噬菌斑与周围红色细菌生长区,颜色差异明显,非常便于观察和计数;噬菌体头部大小和形态与呼吸道病毒中的呼肠病毒和腺病毒最为接近;国内尚未见粘质沙雷氏菌噬菌体相关报道.     

Nutrient utilization during biomass and anthocyanin accumulation in suspension cultures of wild carrot cells

The medium used for the growth of anthocyanin-accumulating wild carrot (D. carota) suspension cultures contained ammonia as a sole nitrogen source and was buffered with succinate. Ammonia was the first nutrient to be completely utilized.The uptake of carbohydrate, phosphate and succinate continued after ammonia depletion. Biomass accumulation was faster and greater when sucrose was initially present in the medium than when glucose was present. When sucrose was provided in the medium it was rapidly hydrolysed to glucose and fructose and the fructose was used preferentially to glucose. Anthocyanin accumulation was rapid after ammonia fell below 3 mM and until the pH of the medium rose from 4.5 to 5.1 or 5.2.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Spectra of chloroperoxidase compounds II and III

Chloroperoxidase was present as Compound II during the peroxidatic oxidation of ascorbic acid. Compound III (oxy-form) was formed when excess hydrogen peroxide was added to Compound II. By decreasing the temperature it was possible to measure the spectra of Compounds II and III in the Soret and visible regions. Each spectrum was found to resemble that of the corresponding form of lactoperoxidase. Under the experimental conditions, chloroperoxidase Compound III was apparently converted to Compound II in parallel with the decomposition of hydrogen peroxide and finally to the ferric enzyme.     

Penile paralysis and paraphimosis associated with reserpine administration in a stallion

Reserpine was administered to an 8-yr-old Thoroughbred stallion at a dosage of 5 mg subcutaneously (s.c.) every 2 wk for a 2-mo period to control unmanageable behavior. Reserpine produced a satisfactory calming effect that lasted for about 2 wk. After the last injection, the stallion developed penile paralysis and was unable to retract his penis, resulting in paraphimosis and attendant penile edema. The prolapsed penis was reduced and kept within the prepuce by placing a purse string suture in the preputial orifice. Phenylbutazone was given orally (1 gm) and the stallion was exercised for 20 to 30 min twice daily. This treatment was continued for 20 d with little improvement. The purse string retention suture cut through the skin 5 d after the stallion was discharged from the clinic. The penis was then supported against the abdominal wall and the stallion was exercised by hand for 30 min each day. The stallion was not used for breeding within 34 mo after the last injection of reserpine. A breeding soundness examination was performed approximately 3 yr after the initial injury. At this time the stallion's penis was noted to extend 5 to 8 cm from the prepuce when in a detumescent state. Although the stallion protruded his penis when exposed to a mare in estrus, a full rigid erection was never attained. Examination of the penis revealed partial engorgement of the corpus cavernosum penis and a 2.5-cm-wide dorsal semi-circumferential depression of the penile shaft approximately 10 to 12 cm proximal to the glans penis. The penile shaft and glans penis distal to this depression were cooler than the proximal portion of the penis. Semen collection was attempted, aided by manual insertion of the penis into the artificial vagina. When serving the artificial vagina, no "belling" of the glans penis was observed, although ejaculation occurred. Semen evaluation indicated normal spermatozoal motility and morphology parameters. The stallion was able to breed several mares with manual assistance to guide the penis into the vagina and one mare was diagnosed pregnant.     

Some characteristics of the insulin-induced potentiation to anaphylactoid reaction in Sprague-Dawley rats.

The insulin-induced sensitization to generalized and local anaphylactoid reaction evoked by dextran was studied in Sprague-Dawley CFY rats. The generalized reaction was shown to be potentiated by insulin given subcutaneously in a dose-related manner. The minimum effective dose was as low as 0.04 U/kg. When this dose was injected intravenously, a marked but short-lived potentiation was observed. The insulin response could be elicited throughout the whole year. The local oedema induced by subplantar injection of dextran was found to be much less sensitive to insulin. Potentiation was observed during the period from March to October, while in the intermediate months, no such effect could be seen. The seasonal refractory state to insulin was abolished by bilateral adrenalectomy, and daily pretreatment of the rats with insulin for several days. Actinomycin D prevented the restorative effect of insulin pretreatment. Sensitization by a single insulin dose to both systemic and local dextran was suppressed in rats older than 6 months, and the refractoriness was in part reversed by adrenalectomy.     

Exospore formation in Methylosinus trichosporium.

Formation of exospores in Methylosinus trichosporium was examined by electron microscopy; serial sectioning was used to visualize the shape and location of the developing exospore in relation to the vegetative cell. The initial stage was the formation of a budlike enlargement on one end of the vegetative cell. The enlargement was surrounded by the exospore capsule, and the cell wall was continuous around both the cell and the developing exospore. A constriction occurred in the area where the budlike structure was attached to the vegetative cell, and the constriction continued to form until the immature exospore was detached from the vegetative cell. The cup-shaped immature exospore was surrounded by the exospore capsule, which appeared to hold the exospore close to the vegetative cell. After separation from the vegetative cell, the immature exospore developed further by forming the exospore wall and by becoming spherical.