Growth of cultured cells from patients with Fanconi anemia

Fibroblast cultures derived from skin biopsies of patients with Fanconi anemia had doubling times (mean of five lines: 30.3 ± 0.2 hours) significantly longer than randomly selected normal controls (mean of nine lines: 22.9 ± 0.4 hours). Control cultures grew more slowly in the enriched media RPMI 1640 and McCoy's 5A than in MEM, while a culture from a patient with Fanconi anemia grew more slowly only in McCoy's 5A. Differences in growth characteristics between Fanconi anemia and normal cell cultures may be useful in analyzing the metabolic error determined by the Fanconi anemia gene.     

实时荧光定量RT-PCR在小鼠及人G蛋白mRNA检测中的应用

用自行设计的TaqMan双标探针和扩增引物建立检测鸟苷酸结合蛋白(G-protein)mRNA的实时荧光定量RT-PCR技术.用G蛋白纯品绘制定量标准曲线,实时荧光定量PCR仪检测ECV304细胞和小鼠G蛋白mRNA水平.10μmol/LGqαmRNA反义寡核苷酸(GqαODN)作用ECV304细胞24h后,Gqα的mRNA表达显著下降(3.18×108±1.75×108拷贝下降到1.44×106±4.82×105拷贝),48h和72h下降更明显;Gsα和Gi2α的mRNA表达变化不显著.10μmol/LGsαmRNA反义寡核苷酸(GsαODN)作用ECV304细胞24h后,Gsα的mRNA表达显著下降(2.97×108±2.68×107拷贝下降到4.16×106±2.00×106拷贝),48h和72h下降更明显;Gqα、Gi2α表达变化不显著.小白鼠油酸致伤后6h,GqαmRNA表达显著下降(1.16×108±8.73×106拷贝下降到3.30×106±1.68×106拷贝),24h下降更显著(9.32×107±1.47×107拷贝下降到4.14×106±1.67×106拷贝);Gsα和Gi2α表达变化的趋势同GqαmRNA.结果准确可靠,重现性好.说明建立的实时荧光定量RT-PCR方法成功地实现了对ECV304细胞和小白鼠肺组织G蛋白不同亚型不同丰度基因表达的检测.     

Leptin Response to Glucocorticoid Occurs at Physiological Doses and Is Abolished by Fasting

Objective: To examine the effects of graded doses of hydrocortisone (HC) on leptin secretion, and determine the effect of fasting. Research Methods and Procedures: This was a randomized, placebo‐controlled, crossover study, with a 1‐week “washout” period between interventions. Eight healthy subjects [age = 36 ± 2.3 years (±SE), body mass index = 31.5 ± 1.6 kg/m²] completed the dose‐response study in which an intravenous infusion of saline (placebo) or HC (30 or 100 mg) was administered for 24 hours. Four healthy subjects (age = 35.2 ± 3.0 years, body mass index = 27.1 ± 2.1 kg/m²) completed the fasting study, which entailed continuous infusion of saline, HC (300 mg/24 hours) in the fed state, or HC (300 mg/24 hours) with total caloric deprivation for 24 hours. Blood sampling was performed every 1 to 2 hours for measurement of leptin, cortisol, insulin, and glucose levels. Results: Peak hyperleptinemia occurred after 16 hours of HC infusion; peak/baseline leptin levels were 129% (placebo), 140% (30 mg of HC for 24 hours, p = 0.05), and 185% (100 mg of HC for 24 hours, p < 0.01). During infusion of HC (300 mg/24 hours or placebo), the peak/baseline plasma leptin levels were 16.1 ± 5.8/12.8 ± 5.9 ng/mL (placebo with food, 126%), 14.6 ± 6.0/12.5 ± 6.5 ng/mL (HC fasting, 117%), and 32.5 ± 12.5/12.0 ± 8.4 ng/mL (HC with food, 271%, p < 0.001). Discussion: Leptin secretory responses occur at physiological doses of HC, are obliterated by fasting, and thus may be of metabolic significance.     

不同剂量异氟醚对中年小鼠认知功能及海马MBP和pNF-H表达的影响

目的:探讨异氟醚对中年小鼠认知功能及海马髓磷脂碱性蛋白(MBP, myelin basic protein)和磷酸化神经丝重亚单位(pNF-H,phosphorylated neurofilament heavy chain)表达的影响。方法:给予中年小鼠不同浓度的异氟醚处理,实验分为对照组和异氟醚处理组(0.5ISO,1.0ISO,1.5ISO),其中异氟醚组小鼠细胞分别给予0.5 MAC,1.0 MAC和1.5 MAC三个浓度的异氟醚处理4小时,对照组给予O_2处理4小时,随后通过水迷宫测试检测其学习记忆能力变化,通过免疫荧光检测海马形态结构及髓鞘相关蛋白MBP和pNF-H表达变化。结果:与对照组相比,(1)类临床浓度的异氟醚处理不影响中年小鼠的自发运动能力(总运动距离:sham:7275.17±1732.58; 0.5ISO:8057.58±1732.58; 1.0ISO:7540.98±1401.61; 1.5ISO:8243.79±1257.65;运动速度:sham:116.75±22.35; 0.5ISO:135.45±32.84; 1.0ISO:130.16±21.38; 1.5ISO:142.31±20.58),但1.0 MAC和1.5 MAC的异氟醚处理明显降低了中年小鼠在水迷宫目标象限的活动时间百分比(sham:58.62±13.70; 0.5ISO:48.92±7.22; 1.0ISO:31.23±13.16; 1.5ISO:30.29±15.76)(P 0.05),且高浓度异氟醚作用强于低浓度异氟醚;(2) 1.0 MAC和1.5 MAC的异氟醚处理明显下调了海马的MBP和p NF-H表达(MBP:sham:60.48±8.20; 0.5ISO:56.69±7.86; 1.0ISO:40.15±4.50; 1.5ISO:31.66±5.46; pNF-H:sham:62.23±9.45; 0.5ISO:55.47±6.98; 1.0ISO:40.16±6.97; 1.5ISO:30.94±5.89)(P 0.05),造成了小鼠海马髓鞘结构损伤,且高浓度损伤强于低浓度。结论:异氟醚可能通过下调中年小鼠海马MBP和pNF-H表达,破坏海马髓鞘完整性而损伤小鼠的学习认知能力。     

Increased resting energy expenditure after 40 minutes of aerobic but not resistance exercise

Objective: Resting energy expenditure (REE) is increased 24 hours after high‐intensity aerobic exercise lasting 60 minutes, whereas results have been inconsistent after resistance training and aerobic exercise of shorter duration. The objective of the study was to compare the effects of 40 minutes of high‐intensity aerobic vs. resistance exercise on REE 19 to 67 hours after exercise. Research Methods and Procedures: REE was compared 19, 43, and 67 hours after 40 minutes of aerobic training (AT; 80% maximum heart rate) or resistance training (RT; 10 repetitions at 80% maximum strength, two sets and eight exercises). Twenty‐three black and 22 white women were randomly assigned to AT, RT, or no training (controls). Exercisers trained 25 weeks. REE was measured after a 12‐hour fast. Results: There was a significant time × group interaction for REE when adjusted for fat‐free mass and fat mass, with post hoc tests revealing that the 50‐kcal difference between 19 and 43 hours (1310 ± 196 to 1260 ± 161 kcal) and the 34‐kcal difference between 19 and 67 hours (1310 ± 196 to 1276 ± 168 kcal) were significant for AT. No other differences were found, including RT (19 hours, 1256 ± 160; 43 hours, 1251 ± 160; 67 hours, 1268 ± 188 kcal). Urine norepinephrine increased with training only in AT. After adjusting for fat‐free mass, REE Δ between 19 and both 43 and 67 hours was significantly related to urine norepinephrine (r = 0.76, p < 0.01 and 0.68, p < 0.03, respectively). Discussion: Consistent with findings on longer duration AT, these results show that 40 minutes of AT elevates REE for 19 hours in trained black and white women. This elevation did not occur with 40 minutes of RT. Results suggest that differences are, in part, due to increased sympathetic tone.     

ERK信号通路对病毒性心肌炎心肌细胞CAR表达的影响

目的:观察细胞外信号调节激酶(Extracellular Regulated Protein Kinases,ERK1/2)信号通路对病毒性心肌炎(Viral Myocarditis,VMC)心肌细胞柯萨奇病毒-腺病毒受体(Coxsackie-adenovirus Receptor,CAR)表达的影响。方法:新生SD大鼠心肌细胞体外培养48 h后随机分为3组,除对照组外均体外接种柯萨奇B3m病毒(Coxsackievirus B,CVB),建立VMC细胞模型。C组:DMEM对照组;V组:CVB3m感染组;U+V组:接种病毒前30 min,给予ERK1/2通路抑制剂U0126(10μmol/L)。各组分别于种毒后12 h、24 h、36 h取心肌细胞,用Western blot法测定ERK1/2活化水平及CAR表达量,并按上述时间点观察各组心肌细胞形态、搏动情况、细胞损伤程度,取培养液测定乳酸脱氢酶(LDH)水平。结果:在接种病毒后12 h,V组与C组相比,P-ERK1/2表达增高(3.25±0.61 vs 0.59±0.09,P0.05),CAR表达增高(1.03±0.17 vs 0.78±0.11,P0.05),逐步出现细胞病变,细胞搏动停止,培养液中LDH水平明显增高(1016.67±67.75 vs 336.34±28.67,P0.05),心肌酶学的升高与镜下心肌细胞损伤程度平行;U+V组与V组相比,P-ERK1/2表达降低(1.66±0.28 vs 3.25±0.61,P0.05),CAR表达明显增高(1.73±0.27 vs 1.03±0.17,P0.05),但细胞损伤却明显减轻,LDH水平明显降低(410.06±13.62 vs 1016.67±67.75,P0.05)。动态观察24 h、36 h,同样出现上述变化趋势。结论:ERK1/2信号转导通路参与心肌细胞感染CVB发生急性损伤的过程,并参与调控CAR的表达。在病毒感染后36 h内,阻断ERK1/2信号通路,CAR表达上调,并未加重心肌细胞损伤。     

Basal and Stimulated Plasma Leptin in Diabetic Subjects

LIU, JIANMEI, HASAN ASKARI, AND SAMUEL DAGOGO-JACK. Basal and stimulated plasma leptin in diabetic subjects. Obes Res. Objective: To determine whether leptin secretion is impaired in diabetes, we compared basal and stimulated plasma leptin levels in diabetic subjects and healthy controls. Research Methods and Procedures: Blood samples for assay of leptin and other hormones were obtained at baseline in 54 diabetic patients and 65 controls, and 8 hours, 16 hours, and 40 hours following ingestion of dexamethasone (4 mg) in 6 healthy and 12 controls. C-peptide status was defined as “negative” if ≤0.1 ng/mL or “positive” if ≥0.3 ng/mL, in fasting plasma. Results: Basal plasma leptin levels were 19. 7±2. 2 ng/mL in nondiabetic subjects, 13. 4±1. 5 ng/ml in C-peptide negative (n = 28) and 26. 1±23. 7 ng/mL in C-peptide positive (n = 26, p = 0. 001) diabetic patients. Dexamethasone increased leptin levels of controls (n = 6) to 145±17% of baseline values at 8 hours (p = O. O3), 224±18% at 16 hours (p = 0. 01), and 134218% at 40 hours (p = 0. 05). The corresponding changes were 108±13%, 126±23%, and 98±16% in C-peptide negative (n = 6), and 121±10%, 144±16% (p = 0. 03), and 147±23% (p = 0. 11) in C-peptide positive (n = 6) diabetic patients, respectively. The peak stimulated leptin levels were lower in the diabetic patients, compared with controls. Plasma insulin increased (p = 0. 02) in controls, but not in the diabetic patients, following dexamethasone. Discussion: Although diabetic patients have normal plasma leptin levels under basal conditions, their leptin responses to glucocorticoid are impaired, probably because of the concomitant insulin secretory defect. A subnormal leptin secretory response could worsen obesity and insulin resistance in diabetes.     

Daily variations in the thermoregulatory behaviors of naked neck broilers in an equatorial semi-arid environment

The aim of this study was to evaluate the daily variations in the thermoregulatory behavior of 4- to 6-week-old naked neck broilers (Label Rouge) in an equatorial semi-arid environment. A total of 220 birds were monitored for 5 days starting at 0600 hours and ending at 1800 hours. The period of observation was divided into classes of hours (C _H). The observed behaviors were as follows: feed and water intake, wing-spreading, sitting or lying, and beak-opening. A total of 14,300 behavioral data values were registered. In C _H 2 (0900 hours to 1100 hours) and 3 (1200 hours to 1500 hours), the greatest average body surface temperature was recorded (34.67?±?0.25 °C and 35.12?±?0.22 °C, respectively). The C _H had an effect on the exhibition of all behaviors with the exception of the water intake behavior. Feed intake was more frequent in C _H 1 (0600 hours to 0800 hours) and 4 (1600 hours to 1800 hours). In C _H 2 and 3, the highest frequency of sitting or lying behavior was observed. Beak-opening and wing-spreading behaviors occurred more frequently in C _H 3 where the body surface temperature (35.12?±?0.22 °C), radiant heat load (519.38?±?2.22 W m^?2), and enthalpy (82.74?±?0.36 kJ kg^?1 of dry air) reached maximum recorded averages. Thus, it can be concluded that naked neck broilers adjust their behavior in response to daily variations in the thermal environment. Wing-spreading and beak-opening behaviors are important adaptive responses to the thermal challenges posed by the equatorial semi-arid environment.     

Diurnal variations of androgens in sexually mature male Bolivian squirrel monkeys (Saimiri sciureus) during the breeding season

To assess diurnal fluctuations of serum androgens and cortisol in adult male Bolivian squirrel monkeys, these steroids were measured at predetermined times (0300, 0900, and 2300 hours) during two separate 24-hour periods in the breeding season (January 1983 and late November 1983). A significant diurnal change in serum cortisol was noted, with a nadir of 99.9 ± 11.9 μg/dl (x? ± SEM) at 2300 hours and a peak of 168.9 ± 7.8 μg/dl at 0900 hours. Conversely, a nadir in serum testosterone was noted at 0900 hours (117 ± 26.5 ng/ml) increasing to a peak of 328.5 ± 57.9 ng/ml at 0300 hours. Serum androstenedione and dehydroepiandrosterone followed a pattern similar to testosterone, with a serum androstenedione (176.4 ± 34.9 ng/ml) and dehydroepiandrosterone (11.7 + 1.8 ng/ml) nadir at 0900 hours and a plasma androstenedione (494.5 ± 55.4 ng/ml) and dehydroepiandrosterone (32.5 ± 4.1 ng/ml) peak at 0300 hours. Parallel changes of testosterone, androstenedione, and dehydroepiandrosterone suggest a significant contribution of all three androgens from a common site, the testes. In contrast to old world primates and humans, serum androstenedione levels exceeded serum testosterone levels in this species.     

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.     

Effect of Dexamethasone on Oral Glucose Tolerance in Healthy Adults

ObjectiveTo determine the dose-response and time course of action of a single dose of dexamethasone on plasma glucose and insulin dynamics in healthy adults.MethodsParticipants included healthy adults who met the following inclusion criteria: 18 to 65 years of age, body mass index of 18 to 25 kg/m2, no family history of diabetes mellitus, not taking any medication known to affect glucose tolerance, and nonpregnant state for female participants. Each participant underwent 3 sequential blocks of 75-g oral glucose tolerance tests (OGTTs) on days 1, 2, and 3; this sequence was repeated on 3 different occasions separated by more than 2 weeks. On the first day of each block, participants reported to the research center after a 10- to 12-hour overnight fast, and fasting baseline blood samples for glucose, insulin, and C-peptide were obtained. Baseline (0 mg) OGTT was then performed with a 75-g glucose load, and blood samples were collected at 30, 60, 90, and 120 minutes for measurements of glucose, insulin, and C-peptide. After the baseline OGTT on day 1, a single dose of either 2-, 4- or 8-mg of dexamethasone was administered orally. Twenty-four and 48 hours later, participants returned for additional OGTTs.ResultsTen healthy volunteers (4 male and 6 female) were enrolled. The effect of dexamethasone was maximal 24 hours after 8-mg dexamethasone compared with the effect observed after no dexamethasone administration. At 60 minutes during the OGTT (following 8-mg dexamethasone), blood glucose increased from 127 ± 7.1 mg/dL (6.35 ± 0.36 mmol/L) to 176 ± 19 mg/dL (8.8 ± 0.95 mmol/L), insulin increased from 49.3 ± 3.2 μIU/mL (342 ± 22 pmol/L) to 119.7 ± 10.1 μIU/mL (831 ± 70 pmol/L), and C-peptide increased from 6376 ± 510 pg/L (1913 ± 153 pmol/L) to 10 143 ± 1016 pg/L (3043 ± 305 pmol/L); the 60-minute levels returned towards baseline at 48 hours. Smaller changes were observed with 2- and 4-mg dexamethasone. Twenty-four hours after 8-mg dexamethasone, there was a 2.2- and 1.5-fold increase in homeostasis model assessment of insulin resistance and homeostasis model assessment of β cell, respectively, and a 2.5-fold decrease in the Matsuda sensitivity index.ConclusionsA single oral dose of 8-mg dexamethasone increases blood glucose, insulin, and C-peptide levels maximally at 24 hours, 1 hour following 75-g OGTT. A dexamethasone stress test might identify persons at increased risk for type 2 diabetes. (Endocr Pract. 2010:16:770-777)     

后腹腔镜保留肾单位手术治疗早期肾癌患者的术肾肾功能分析

目的:探讨后腹腔镜保留肾单位手术(LNSS)对早期肾癌患者术后术肾肾功能的影响。方法:收集并随访新疆维吾尔自治区人民医院泌尿外科2009年1月~2012年6月接受经后腹膜行腹腔镜保留肾单位术治疗,且术后病检结果为肾癌患者的临床资料,分别于术前、术后24小时、2周、6月、1年、1.5年、2年测定双肾GFR、血清肌酐、血清胱抑素指标值,随访时间大于2年的有28例患者,比较并分析各指标值的变化情况,分析LNSS术对肾功能的影响。结果:28例患者术肾术前GFR及占总GFR的比例分别为42.02±7.31 ml/min和43.30±3.6%,术后2周分别为31.42±5.23 ml/min和34.83±5.8%,术后6月分别为33.23±5.46ml/min和36.85±5.3%,术后1年分别为37.21±6.59 ml/min和39.74±6.2%,术后1.5年分别为40.44±5.82 ml/min和42.26±6.2%,术后2年分别为40.64±5.74 ml/min和42.26±5.8%。术后24小时,血清肌酐水平升高,术后6个月以后与术前比较无明显差别。术后2周,血清胱抑素水平升高,术后6个月恢复到术前水平。结论:LNSS术式对早期肾癌是安全有效的。     

长链非编码RNA对HepG2 肝癌细胞增殖及凋亡的影响

目的:探讨长链非编码RNA RP13-514E23.1在肝癌细胞系中的表达,并观察上调该基因后其下游基因MAPK10的变化及对肝癌细胞Hep G2增殖、凋亡的影响。方法:通过荧光定量PCR(q RT-PCR)检测RP13-514E23.1在正常肝细胞与肝癌细胞系中的表达差异,进一步构建质粒转染肝癌Hep G2细胞上调RP13-514E23.1的表达,以Mock组(只加转染试剂)和NC组(转染空载体pc DNA3.1-NC)作为对照评估转染效率及对MAPK10表达的影响。用CCK-8实验和流式细胞术检测转染前后Hep G2细胞的增殖、凋亡的变化。结果:Lnc RNA RP13-514E23.1在大部分肝癌细胞系(Hep G2,SMMC,Huh7,Hep3B)中的表达明显低于正常肝细胞(0.58±0.05 vs 1.00,P0.05);转染pc DNA-RP13-514E23.1后,q RT-PCR检测Hep G2细胞的RP13-514E23.1和MAPK10m RNA表达量显著升高(分别为29.90±1.40、2.42±0.25,P0.05),western blot检测MAPK10蛋白表达量较对照组也升高(2.10±0.16,P0.05);CCK-8结果显示Hep G2细胞在各个时间段增殖均受到抑制(P0.01),Mock组、NC组和实验组的凋亡率分别为(5.53±1.17)%、(6.40±2.84)%和(46.87±3.45)%(P0.01)。结论:Lnc RNA RP13-514E23.1在肝癌细胞系中表达异常降低,上调其表达后MAPK10的表达升高,且Hep G2细胞增殖受到抑制、凋亡增加。     

Effects of lighting programs on onset of the ovulatory season in mares

Using 240 pony mares, lighting regimens were tested for their efficiency in hastening the onset of the ovulatory season. The mean number of days from January 1 to first ovulation was used as the end point. No advantage was gained by beginning a fixed lighting regimen ($\text{15.5L}\text{8.5D}$, hours light/hours dark) November 1 (66 ±8) versus December 1 (65 ±9), but beginning on January 1 was less efficient (98 ±8; controls, 132 ±5; P<0.05). In another experiment, daily three-hour interruptions of either the light phase (67 ±10) or the dark phase (71 ±11) did not significantly retard the effectiveness of a fixed regimen of $\text{15L}\text{9D}$ (54 ±5; controls, 142 ±6). A $\text{15L}\text{9D}$ regimen every other day (natural day length on alternate days) resulted in an interval (85 ±7) that was shorter (P<0.05) than for the controls and longer (not significant) than for the daily $\text{15L}\text{9D}$ regimen. When used with natural day length, a one-hour pulse of light in the evening (15 hours after sunrise) was not effective (141 ±6); a one-hour pulse in the morning 9.5 hours after sunset) was only partially effective (117 ±6). In another experiment, the interval was reduced (P<0.05) in a group with one hour of light fixed at 4:00 a.m. with natural day length (85 ±8; $\text{15L}\text{9D}$, 75 ±7; controls, 126 ±9). Results indicated that a fixed one-hour pulse of light at 4 a.m., used with natural day length, may provide an acceptable level of stimulation.     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Pulmonary absorption of liposomal levonorgestrel

The purpose of these studies was to achieve desired bioavailability after pulmonary administration of Levonorgestrel (LN) and to provide prolonged effective concentration of the drug in plasma and to reduce reported side effects of orally administered drug. The plain drug suspension, physical mixture (plain drug with liposomal constituents), and drug-encapsulated liposomes containing 10 μg of drug were instilled intratracheally in rats. Similarly, 10-μg drug suspension (LO) was administered orally. The blood samples were withdrawn at specific time intervals and were subjected to LN analysis by spectrofluorimetric technique. The plasma drug concentration data of both the treatments were plotted, and pharmacokinetics data were calculated and compared with that of oral administration. Percentage relative bioavailability (F^*) of 97.6% 98.6%, and 109.9% were observed after pulmonary administration of plain drug formulation (LP1), physical mixture (plain drug along with constituents of liposomes [LP2], and liposomal (LP3) formulations of the drug, respectively. Following oral administration, C_max of 14.4±0.6 ng/mL was observed at 2.1±0.2 hours followed by subtherapeutic concentration beyond 30±0.2 hours, while after pulmonary administration of LP1, LP2, and LP3 formulations, C_max of 4.4±0.4 ng/mL, 4.2±0.5 ng/mL, and 4.4±0.6 ng/ML were observed at 6.0±0.2 hours, 7.0±0.2 hours, and 6.8±0.2 hours, respectively, followed by maintenance of effective plasma drug concentration up to 60±2 hours. These studies demonstrate superiority of pulmonary drug delivery with regards to maintenance of effective therapeutic concentration of the LN in the plasma over a period of 6 to 60 hours. Hence, the pulmonary delivery is expected to reduce frequency of dosing and systemic side effects associated with oral administration of LN.     

Luteinizing hormone concentrations in anestrous ewes administered various estrogens

Twenty four anestrous ewes were evenly assigned to one of six groups and administered either sesame oil, estradiol-17β, estradiol-17α, estrone, estradiol benzoate or estradiol valerate. All estrogen treated ewes received 50 μg of the respective estrogen. Blood plasma was collected for 28 hours post-treatment and quantified for luteinizing hormone (LH) by radioimmunoassay. An estrogen induced LH surge was detected in at least three of the four ewes administered either estradiol-17β, estrone, estradiol benzoate or estradiol valerate whereas only one of the four estradiol-17α treated ewes and none of the ewes administered sesame oil had an LH surge. The interval from treatment to peak LH was similar for estradiol-17β (17.3±2.7 hours), estrone (18.5±1.0 hours) and estradiol benzoate (19.0±0.6 hours) treated ewes but delayed 7 to 9 hours for ewes administered estradiol valerate (26.0±1.2 hours).     

Transdermal enhancement through rat skin of luciferase plasmid DNA loaded in elastic nanovesicles

Transdermal absorption of luciferase plasmid (pLuc) was enhanced by loading in elastic cationic liposomes and niosomes and the application of iontophoresis or the stratum corneum (SC) stripping method. Cationic liposomes (DPPC/Chol/DDAB at a 1:1:1 molar ratio) and niosomes (Tween61/Chol/DDAB at a 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes method. The elastic vesicles were prepared by hydrating the lipid or surfactant film by 25% of ethanol instead of distilled water. Gel electrophoresis of all nanovesicles showed the 100% pLuc entrapment efficiency. All nanovesicles loaded with pLuc showed larger vesicular sizes than the nonloaded vesicles of about 1.4 times for liposomes and 1.7 times for niosomes. The nanovesicles loaded with pLuc demonstrated less positive zeta potential than the nonloaded vesicles. The pLuc loaded in elastic vesicles kept at 4 ± 2 and 27 ± 2°C for 8 weeks gave the remaining pLuc of about 70 and 60% for liposomes and 85 and 73% for niosomes, respectively. For nonelastic vesicles kept at 4 ± 2°C, 56 and 61% of the remaining pLuc were observed for liposomes and niosomes, respectively, while at 27 ± 2°C, all pLuc were degraded. The deformability indices of the elastic liposomes and niosomes loaded with the pLuc were 16.64 ± 2.92 and 20.72 ± 0.82, whereas the nonelastic vesicles gave 9.35 ± 0.09 and 10.08 ± 0.12, respectively. Transdermal absorption through rat skin pretreated with SC stripping or treated with iontophoresis of pLuc loaded in nanovesicles by vertical Franz diffusion cells was investigated at 37°C. The cells were stopped and the skin and the receiving solution were withdrawn at 1, 3, and 6 hours and the pLuc contents in the stripped SC, whole skin (viable epidermis and dermis; VED), and the receiving solution were assayed by the modified gel electrophoresis and gel documentation. Without the SC stripping technique or iontophoresis, the pLuc loaded and nonloaded in nonelastic cationic liposomes or niosomes were not found in SC, VED, and receiving solution. The fluxes in the whole skin of pLuc loaded in nonelastic liposomes and niosomes with SC stripping and iontophoresis at 6 hours gave 2.73 ± 0.46 and 3.83 ± 0.73, and 7.01 ± 1.22 and 9.60 ± 1.31 g/cm²/h, respectively, while pLuc loaded in elastic liposomes and niosomes without the SC stripping and iontophoresis at 6 hours showed 2.79 ± 0.09 and 2.84 ± 0.04 g/cm²/h, respectively. The pLuc loaded in elastic niosomes or in nonelastic niosomes with iontophoresis was found in the receiving solution with a higher amount than that loaded in elastic liposomes or nonelastic liposomes with iontophoresis. The fluxes in the receiving solution of pLuc loaded in nonelastic liposomes and niosomes with iontophoresis at 6 hours were 6.71 ± 0.31 and 8.82 ± 0.28 g/cm²/h, respectively. For elastic liposomes and niosomes, the fluxes of the loaded pLuc in the receiving solution were the same, at about 1.9 g/cm²/h. Although pLuc loaded in nonelastic niosomes with iontophoresis gave the highest delivery of the plasmid in VED and receiving solution, a more promising applicable approach for gene delivery has been suggested to be the elastic niosomal systems, since no equipment is required.     

Exposure to strong static magnetic field slows the growth of human cancer cells in vitro

Proposals to enhance the amount of radiation dose delivered to small tumors with radioimmunotherapy by constraining emitted electrons with very strong homogeneous static magnetic fields has renewed interest in the cellular effects of prolonged exposures to such fields. Past investigations have not studied the effects on tumor cell growth of lengthy exposures to very high magnetic fields. Three malignant human cell lines, HTB 63 (melanoma), HTB 77 IP3 (ovarian carcinoma), and CCL 86 (lymphoma; Raji cells), were exposed to a 7 Tesla uniform static magnetic field for 64 hours. Following exposure, the number of viable cells in each group was determined. In addition, multicycle flow cytometry was performed on all cell lines, and pulsed-field electrophoresis was performed solely on Raji cells to investigate changes in cell cycle patterns and the possibility of DNA fragmentation induced by the magnetic field. A 64 h exposure to the magnetic field produced a reduction in viable cell number in each of the three cell lines. Reductions of 19.04 ± 7.32%, 22.06 ± 6.19%, and 40.68 ± 8.31% were measured for the melanoma, ovarian carcinoma, and lymphoma cell lines, respectively, vs. control groups not exposed to the magnetic field. Multicycle flow cytometry revealed that the cell cycle was largely unaltered. Pulsed-field electrophoresis analysis revealed no increase in DNA breaks related to magnetic field exposure. In conclusion, prolonged exposure to a very strong magnetic field appeared to inhibit the growth of three human tumor cell lines in vitro. The mechanism underlying this effect has not, as yet, been identified, although alteration of cell growth cycle and gross fragmentation of DNA have been excluded as possible contributory factors. Future investigations of this phenomenon may have a significant impact on the future understanding and treatment of cancer. © 1996 Wiley-Liss, Inc.     

Overexpression of a Catharanthus tryptophan decarboxylase (tdc) gene leads to enhanced terpenoid indole alkaloid (TIA) production in transgenic hairy root lines of Rauwolfia serpentina

To enhance the production of terpenoid indole alkaloids in Rauwolfia serpentina, Catharanthus tryptophan decarboxylase (Crtdc) gene was over-expressed in transgenic hairy root cultures using Agrobacterium rhizogenes-mediated transformation. Among six transgenic hairy root lines, line RT4 accumulated the highest alkaloid content, with 0.1202 % dry weight (DW) reserpine and 0.0064 % DW ajmalicine, after 10 weeks of culture. Whereas, wild-type roots accumulated 0.0596 ± 0.003 % DW reserpine and 0.0011 ± 0.001 % DW ajmalicine. Transgenic hairy root line RT7 produced the lowest alkaloid content (reserpine: 0.0896 ± 0.002 % DW; ajmalicine: 0.002 ± 0.0 % DW). On the basis of alkaloid content the six hairy root lines were grouped as RT4/RT2 > RT3/RT5 > RT7/RT8. Analysis of gene expression profile indicated that Crtdc was expressed at a higher level in transgenic lines, which could be correlated with enhanced metabolite accumulation in roots. This study confirms that over-expression of Crtdc is a superlative method to improve the biosynthetic potential of Rauwolfia hairy root cultures. Enhanced reserpine and ajmalicine production can serve as an alternative choice to provide resources for relative pharmaceutical industries.