Plastic Centrifuge Tube Research Articles

A simulated sunlight-activated plastic mimic enzyme for dual-mode detection of D-penicillamine and serum iron

No reports disclosed that whether the plastic centrifuge tube with intrinsic photoenzyme activity can be used for the specific sensing of pharmaceuticals and biomarkers in biofluids so far. We herein report the plastic eppendorf (EP) tube as both reaction vessel and sunlight-activated plastic mimic enzyme for colorimetric/smartphone dual-mode detection of D-penicillamine (D-PA) and iron (Ⅲ) ions in goat and rabbit serums accordingly. The photooxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) combined with simulated sunlight-activated EP tube was selectively inhibited by adding D-PA, and the absorbance at 652 nm of oxidized TMB decreased. Good linearity between the absorbance variation (or R/(R + G + B)) and D-PA level ranging from 10 to 300 µM was obtained, and the limits of detection (LOD) were determined as 8.50 μM (colorimetric) and 6.92μM (smartphone). Furthermore, the polyvinyl alcohol hydrogel encapsulated TMB in EP tube as a smartphone biosensor had been triumphantly used to monitor D-PA level in goat serum. Interestingly, the adsorption band of photo-oxidized EP tube-TMB system inhibited by D-PA gradually increased as the enhancement of iron (Ⅲ) ions concentration. Based on the “on–off-on” effect, a sensitive and selective method was further used for selectively sensing iron (Ⅲ) in rabbit serum. Notably, the dual-mode sensing platforms exhibited outstanding detection performance, with LOD values as low as 0.50 µM and 0.65 µM respectively. In short, this work not only excavated a photoactivated-plastic mimics, but also developed a robust, low-cost, simple and sensitive biosensor for simultaneous detection of D-PA and Fe (Ⅲ) ions in biofluids. The current photoactivated sensor does not requires the introduction of exogenous catalyst except for simulated sunlight, plastic tube and chromogenic substrate.

Simultaneous determination of 36 hypotensive drugs in fingerprints by ultra performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry

指印中蕴含着供体摄入成分等相关信息,通过对其分析可对供体进行特征刻画,从而为案件侦查提供线索,指印分析也可用于药物摄入的定性检测,因此检验指印中的降压药具有重要的实际应用价值。建立了超高效液相色谱-三重四极杆复合线性离子阱质谱(UPLC-Q-TRAP/MS)同时检测指印中36种降压药的方法。前处理方法采用沉淀蛋白法,使用3×3 cm滤纸采集指印,将滤纸剪碎置于2 mL塑料离心管中,加入0.50 mL甲醇,涡旋混合1 min,超声振荡3 min,取出后以12000 r/min离心5 min,取上清液进样分析。采用ACQUITY UPLC® BEH C18柱(100 mm×3.0 mm, 1.7 μm)分离,以0.01%甲酸水溶液和甲醇作为流动相进行梯度洗脱。质谱分析采用可编程多反应监测-信息关联采集-增强子离子(SMRM-IDA-EPI)扫描方式,在高灵敏度分析的同时进行二级谱库检索,增加定性结果的准确性。36种药物在0.05~50.00 ng/fingerprint范围内线性关系良好,相关系数(r)大于0.99,检出限和定量限分别为0.001~0.045 ng/fingerprint和0.002~0.050 ng/fingerprint,在0.25、2.50、25.00 ng/fingerprint 3个加标水平下的基质效应为79.0%~119.2%,回收率为79.3%~116.2%,日内精密度为0.2%~18.3%,日间精密度为1.6%~19.1%。使用该方法检测了87名高血压患者指印中的降压药,大部分样本中可检出其所服用的药物。该方法操作简单,灵敏度高,选择性好,适用于指印中降压药的筛查检验。

On Language, Scientific Metaphor, and Endocrine Disruption: An Interview with Feminist Scientist Ana Soto

In the late 1980s, Professor of Immunology Ana Soto accidentally discovered the presence of synthetic estrogens in her lab equipment. Her lab had designed an experiment to test the effect of estrogen on the proliferation of human breast cancer cells (MCF7). Based on previous findings, Soto and her research partner Carlos Sonnenschein believed that, contrary to popular wisdom, the introduction of estrogen would not directly induce the proliferation of the cells, but would instead interfere with a naturally occurring inhibitor in the blood. But the control setup containing positive and negative controls (used in the past without problem) was now producing odd readings: although no estrogenic compound had been introduced, the cancer cells were still proliferating. Soto and Sonnenschein methodically removed each item in the control setup that might be producing the estrogen-like result. When they discovered that the estrogenic activity leached from the plastic centrifuge tubes used to store components of the cell culture medium, they called the manufacturer to find out what could have changed. The manufacturer let them know that the constituent materials of the tubes had recently been modified in order to reduce the possibility of breakage during centrifugation but declined to reveal what specific changes had been made. So Soto’s lab turned to studying the tubes themselves. After a year of further research, they concluded that the estrogenic activity was due to the additive that had been introduced by the manufacturer—nonylphenol, an antioxidant used in numerous other applications, some of which are meant for human use (e.g., spermicide) and the synthesis of detergents. Soto and Sonnenschein’s assay is called E-SCREEN and has been enormously influential; in fact, most of the environmental estrogens discovered in the 1990s rely on it.
 Conducted by Gracen Brilmyer at UCLA during the Chemical Entanglements Symposium, this 2017 interview with Soto highlights her experiences of sexism in the sciences and how those experiences have shaped her thinking on language, science, and scholarship

Acrosome integrity and mitochondrial membrane potential of frozen thawed buffalo semen treated with heparin binding protein

The experiment was conducted to study the sperm acrosome integrity and mitochondrial membrane potential (MMP) of frozen thawed buffalo semen treated with heparin binding protein (HBP). Buffalo semen straws from 10 bulls were procured from Central Frozen Semen Production and Training Institute, Hesseraghatta, Banglore-560088. The frozen straws were thawed at 37 ºC for 30 seconds and emptied into a 15mL sterile plastic centrifuge tube containing 1mL capacitation medium (control), addition of 25µg/mL (treatment I), 50µg/mL (treatment II) and 100µg/mL (treatment III) of HBP. The contents were incubated at 37 ºC for 2 hours. After incubation, sperm acrosomal integrity was assessed by Giemsa stain method. In control, HBP treatment I, II and III, 51.50% ± 1.29, 46.30% ±0.86, 43.40% ± 0.66and 42.15% ± 0.40 spermatozoa, respectively had intact acrosome. But, 48.50% ±1.29, 53.70% ± 0.86, 56.60% ± 0.66and 57.85% ± 0.40 spermatozoa in control, HBP treatment I, II and III respectively had lost acrosome. Significantly (P

Developing the OECD 106 fate testing protocol for active pharmaceuticals in soil

ABSTRACT The ability to determine accurately the fate of APIs in soil is essential for rigorous risk assessment associated with wastewater reuse or biosolid recycling to land, particularly in lower income countries where water and fertiliser is scarce. Four APIs (naproxen, ofloxacin, propranolol and nevirapine) with wide ranging functionality were used as examples in the development of the OECD 106 soil partitioning and/or degradation study, with naproxen used to illustrate applying the full methodology. The data showed key methodological criteria require careful consideration and testing to generate accurate and consistent results. Only glass fibre membranes were suitable for all APIs, without unduly adsorbing APIs to their surface, thus effectively restricting the minimum practical pore size to 0.7 µm. Polypropylene plastic centrifuge tubes were shown to be suitable, with careful determination of recoveries. Direct injection liquid chromatography-mass spectrometry could reliably resolve all 4 APIs down to less than µg L−1 in soil solutions, although allowance for matrix effects via standard additions was required in some cases. Greatest analytical challenges were found for the highest molecular weight API with the greatest affinity for sorption to surfaces (ofloxacin). Key variables that can impact on partitioning such as solution pH and dissolved organic carbon concentrations were shown to vary within tests over time and should be accounted for.

Determination of fluorine by total reflection X-ray fluorescence in fluoride fluxes

Total reflection X-ray fluorescence spectrometry (TXRF) is known as a state-of-the-art and fast-growing analytical technique. The spectral range of TXRF spectrometry encompasses elements from sodium to uranium. The object of this study was to develop, optimize, and validate the analytical method for fluorine in a form of calcium fluoride determination in fluoride fluxes by total reflection X-ray fluorescence spectrometry. The sample was lixiviated in acetic acid, then, the sample was filtered and the sediment and the filter were ignited at 650 °C. A portion of dried and ignited and weighed sample was placed in a plastic centrifuge tube. To this tube Triton X-100, Ga as internal standard and polyvinyl alcohol were added. The accuracy of the element determinations was estimated by comparison between obtained and certified values of the elements determined in the Certified Reference Materials of fluorite (fluorspar). The limit of detection for TXRF measurement is 4.26 μg F · dm−3 whereas the limit of quantification equals 12.78 μg F · dm−3. The variation coefficient that characterized the reproducibility of the measurement equals 16.94%. In addition to this, it was found that the uncertainty of results associated with the sample preparation process for TXRF measurement is negligible, compared to the measurement uncertainty at the measurement stage using TXRF spectrometry. The results of this work demonstrate the acceptable and fully satisfactory validation criteria such as limit of detection and quantification, accuracy, and precision.

Polyacrylic acid-coated nanoparticles loaded with recombinant tissue plasminogen activator for the treatment of mice with ischemic stroke

Nanoparticle-based thrombolysis is a potential new treatment for stroke. The aim of this study was to investigate the efficacy of targeted thrombolysis using recombinant tissue plasminogen activator (rtPA). The rtPA was covalently bound to magnetic nanoparticles (MNP) and maintained at the target site using an external magnet. Polyacrylic acid (PAA)-coated MNP were synthesized and rtPA was then bound to the resultant PAA-MNP via carbodiimide-mediated amide bonds. For the in vitro tests, blood clots were formed in plastic centrifuge tubes with anti-coagulated plasma, thrombin and calcium chloride. For the in vivo tests, mice with ferric chloride-induced distal middle cerebral artery occlusion were treated with phosphate-buffered saline (PBS), MNP, rtPA, or MNP-rtPA (n = 6 mice per group). The binding efficacy was 80.7 ± 1.5 μg rtPA bound to 1 mg PAA-MNP. In the in vitro tests, the mean lysis percentage dramatically increased from 1.28% in the MNP group without rotation to 77.40% in the rtPA + MNP group with rotating magnetic field. The lysis efficiency of MNP-rtPA was 27.3 ± 1.3%, and it increased to 42.8 ± 2.8% with magnetic field rotation. The mean sizes of the infarct areas of the PBS, MNP, rtPA, and MNP-rtPA mouse groups were 20.09 ± 6.07, 18.28 ± 2.69, 8.65 ± 3.63 and 4.40 ± 2.46 mm3, respectively. Thus, targeted MNP-rtPA accelerated thrombolysis and reduced the infarct area in a mouse model of cerebral embolism. This approach may serve as a feasible and effective treatment for embolic cerebral ischemia.

An efficient rearing system rapidly producing large quantities of poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae), under laboratory conditions

The poultry red mite, Dermanyssus gallinae, is one of the most economically deleterious ectoparasites affecting egg-laying hens in many parts of the world. New approaches to control D. gallinae often require the maintenance of colonies of D. gallinae under laboratory conditions. In the present study, we present an efficient rearing system for D. gallinae, consisting of a metal cage, a plastic storage box and a tray filled with water. Chicks were raised in the cage as host animals. A novel trap was developed to monitor the dynamic changes of mite populations, made with a plastic centrifuge tube and a disposable breathing mask with folds. Mite parameters were analyzed, including number of mites and eggs, survival and feeding rates, oviposition, hatchability and the proportion of D. gallinae at different life stages. The results show that the rearing system had a 53.5-fold increase in the number of mites over a period of six weeks after the introduction of mites. The survival rates of mites were above 94%, and the mean feeding rates ranged from 22.57% to 37.30%. The mean number of eggs per female ranged from 3.42 to 3.50, with the hatchability of eggs above 97%. Nymphs made up most of the population, ranging from 71.46% to 81.37%, while the population of larvae was minor and ranging from 7.54% to 13.04%. The mask trap used in this study was an effective and convenient device to shelter D. gallinae and monitor the dynamic changes of the mite population. The rearing system proved very effective in maintaining and reproducing colonies of D. gallinae, with great potential for the evaluation of the efficacy of vaccines or compounds against D. gallinae under laboratory conditions. It would be a useful tool for close observations in studies on the biology, acology and physiology of poultry red mites.

Hybrid molecularly imprinted polymers synthesized with 3-aminopropyltriethoxysilane-methacrylic acid monomer for miniaturized solid-phase extraction: A new and economical sample preparation strategy for determination of acyclovir in urine

The miniaturized molecularly imprinted solid-phase extraction (mini-MISPE) coupled with high-performance liquid chromatography was proposed for the determination of acyclovir in urine. 1.5-mL tapered plastic centrifuge tube filled with hybrid molecularly imprinted polymers (HMIPs) was used as the cartridge of mini-MISPE, and the HMIPs synthesized with 3-aminopropyltriethoxy silane-methacrylic acid as monomer exhibited good recognition and selectivity for acyclovir. Under the optimized condition, good linear calibration was obtained in a range of 0.5–15μgmL−1 with the correlation coefficient of 0.9994, and the recoveries at three spiked levels were 91.6–103.3% in urine with the relative standard deviation (RSD) of ≤3.5%. Excellent intra-day and inter-day repeatability were achieved with RSD of ≤2.6% and 4.0% in three different concentrations. This method combined the advantages of HMIPs and mini-MISPE, and it could become an alternative tool for analyzing the residues of acyclovir in complex urine matrices.

A Gas Chromatography–Mass Spectrometry Method for the Determination of Pogostone in Canine Plasma and Its Application to a Pharmacokinetic Study

In this study, a simple and selective gas chromatography-mass spectrometry method was developed and validated for the determination of pogostone in canine plasma. Liquid-liquid extraction was used to separate pogostone from canine plasma, and the mean extraction recovery rates of pogostone and the internal standard (isoalantolactone) were 80.61 and 75.89%, respectively. Plastic centrifuge tubes were inadequate for the plasma sample treatment procedure because of the adsorption effect of pogostone on the inner surface. The chromatographic separation was performed on a capillary column of Agilent HP-5ms, and the spectrometer was operated in an electron-impact ionization with an electron multiplier voltage mode. The standard curve was linear over the concentration range of 1.02-406 ng/mL (r > 0.99). The intra- and interday accuracies for pogostone at four concentrations were 97.77-99.92% and 98.51-100.22%, respectively. The relative standard deviations were <15%. The method was successfully applied to a pharmacokinetic study after the oral administration of pogostone to beagle dogs.

In vitro dissolution of uranyl acetate using different methods

The dissolution rate of a material in the lung is an important parameter in evaluating the risk to humans following accidental inhalation of a substance and is also a parameter that may be useful in characterizing particles for nuclear forensics analysis. Conventional methods of measuring dissolution rates in vitro involve exposing the material or particles to a solvent, such as water, saline, or solutions that simulate lung fluid, and measuring the fraction of material that dissolves with time. A new device for measuring dissolution rates for small samples, especially individual particles, was evaluated that incorporates a regenerated cellulose dialysis membrane fixed to the bottom of a small, 2 mL plastic cup that fits into the top of a 50 mL plastic centrifuge tube. The cup is easily transferred among a series of tubes containing solvent to measure rate of dissolution. The dialysis membrane has a diffusion rating of 20 kDa molecular weight cut off which greatly exceeds the size of the dissolved uranium molecule. The performance of the dialysis cup device was evaluated by measuring the dissolution rate of uranyl acetate in distilled water, phosphate buffered saline (PBS), and simulated lung fluid (SLF). These results were compared to the dissolution rate measured using the traditional filter sandwich method in which a sample is sealed between two hydrophilic membranes. Although the majority of uranyl acetate dissolved in SLF within 30 min using the filter sandwich method, most of the uranyl acetate was undissolved in PBS and SLF using the dialysis membrane device. Reactions between the dissolved uranyl acetate, solvent, and the dialysis membrane likely caused the membrane to swell, shrinking the pore size, and thus reducing the transport of dissolved uranium across the membrane. Use of the dialysis cup device for evaluating dissolution rates for uranium-bearing materials in solvents containing a high concentration of salts is therefore not recommended.

Effects of Ultraviolet-A and Riboflavin on the Interaction of Collagen and Proteoglycans during Corneal Cross-linking

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

In vitro penetration of Salmonella Enteritidis through yolk membranes of eggs from 6 genetically distinct commercial lines of laying hens

Although deposition of Salmonella Enteritidis inside yolks is less common than deposition in albumen or on the vitelline (yolk) membrane in naturally contaminated eggs laid by infected hens, bacterial migration into the yolk to reach its nutrient-rich contents could lead to extensive multiplication. The present study used an in vitro egg contamination model to assess the ability of small initial numbers of Salmonella Enteritidis to penetrate the vitelline membrane and multiply inside yolks of eggs laid by 6 genetically distinct commercial lines of hens during 24 h of storage at 30 degrees C. Eggs from each line were tested at 4 different hen ages by inoculation of approximately 100 cfu of Salmonella Enteritidis onto the outside of the vitelline membranes of intact yolks in plastic centrifuge tubes and then adding back the albumen into each tube before incubation. Overall, the frequency of penetration of Salmonella Enteritidis into the yolk contents of eggs from individual lines of hens ranged from 30 to 58% and the mean concentration of Salmonella Enteritidis in yolk contents after incubation ranged from 0.8 to 2.0 log(10) cfu/mL. For both of these parameters, values for one hen line were significantly higher than for 2 other lines, but no other differences were observed. Hen age did not have a significant effect on egg yolk penetration by Salmonella Enteritidis. These results indicate that opportunities for the migration and growth of small initial numbers of Salmonella Enteritidis to attain more dangerous levels inside contaminated eggs during storage at warm temperatures can sometimes vary between different lines of laying hens.

Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen

The aim of this study was to investigate the effects of methionine and dithioerythritol, added to the Tris extender, on ram sperm motility and LPO (lipid peroxidation) and antioxidant capacities during liquid storage up to 72 h at 5 °C. Ejaculates collected from five Merino rams, were evaluated and pooled at 37 °C. This study included two experiments. In experiment 1, each pooled ejaculate was divided into four equal aliquots and diluted (37 °C) with the base extender, containing 0 (control), 1, 2 and 4 mM methionine, at a final concentration of approximately 4 × 10 8 sperms/ml (single step dilution), in a 15-ml plastic centrifuge tube. In experiment 2, dithioerythritol, at concentrations of 0 (control), 0.5, 1 and 2 mM, was used as an additive in the extender, and the procedure explained above was applied for the division of aliquots and the dilution of semen. Diluted semen samples were kept in glass tubes and cooled from 37 to 5 °C in a cold cabinet, and maintained at 5 °C. Sperm motility and LPO and total glutathione (GSH) and glutathione peroxidase (GPx) capacities were determined at 5 °C for periods of 0, 24, 48 and 72 h of liquid storage. The extender supplemented with 1 mM methionine led to higher motility percentages (77.0 ± 1.2%), in comparison to the control group (66.0 ± 4.9%), during 72 h of liquid storage ( P < 0.05). As regards dithioerythritol, it did not statistically improve the motility rates for any of the storage times at 5 °C. In biochemical assays, differences in LPO levels between the groups with antioxidants and the control groups were not statistically significant. Compared to the control group, no significant difference was observed in GSH and GPx activities following the addition of methionine, during 72 h of storage. Total GSH and GPx activities did not increase significantly upon supplementation with 0.5 and 1 mM of dithioerythritol, compared to the control group, at any of the time points ( P > 0.05). Dithioerythritol at 2 mM led ( P < 0.01) to elevating GSH activity, compared to the control group, during 72 h of liquid storage. GPx activity was approximately 10 times higher for 2 mM of dithioerythritol ( P < 0.001), compared to that of the control group at all time points. The question regarding the sustainability of sperm survival, LPO and antioxidant capacities following liquid storage of semen remains unanswered. Further studies are required for a better understanding of the biochemical changes and to obtain more information on the determination of lipid peroxidation and antioxidant capacities during cooled storage of ram semen.

Methane Production by Methanogens Following an Aerobic Washing Procedure: Simplifying Methods for Manipulation

The recent discovery of methane in the martian atmosphere is arguably one of the most important discoveries in the field of astrobiology. One possible source of this methane could be a microorganism analogous to those on Earth in the domain Archaea known as methanogens. Methanogens are described as obligately anaerobic, and methods developed to work with methanogens typically include anaerobic media and buffers, gassing manifolds, and possibly anaerobic chambers. To determine if the time, effort, and supplies required to maintain anaerobic conditions are necessary to maintain viability, we compared anaerobically washed cells with cells that were washed in the presence of atmospheric oxygen. Anaerobic tubes were opened, and cultures were poured into plastic centrifuge tubes, centrifuged, and suspended in fresh buffer, all in the presence of atmospheric oxygen. Washed cells from both aerobic and anaerobic procedures were inoculated into methanogenic growth media under anaerobic conditions and incubated at temperatures conducive to growth for each methanogenic strain tested. Methane production was measured at time intervals using a gas chromatograph. In three strains, significant differences were not seen between aerobically and anaerobically washed cells. In one strain, there was significantly less methane production observed following aerobic washing at some time points; however, substantial methane production occurred following both procedures. Thus, it appears that aerobic manipulations for relatively short periods of time with at least a few species of methanogens may not lead to loss of viability. With the discovery of methane in the martian atmosphere, it is likely that there will be an increase in astrobiology-related methanogen research. The research reported here should simplify the methodology.

Heat-shock Treatments Alter the Kinetics of Ion Leakage from Chilled Tomato Pericarp Tissue

Heat-shock induced chilling tolerance in excised discs of tomato fruit pericarp tissue significantly alter the kinetics of chilling-induced ion leakage from the discs into an aqueous isotonic mannitol solution. Pericarp discs were excised from mature-green tomato fruit, trimmed of locular material to 5-mm thickness, held overnight (ca. 16 h) at 20 °C and then subjected to various heat-shock (45 °C) treatments before being chilled at 2.5 °C for up to 30 days. Two discs were immersed in 20 mL of 0.3 m mannitol in a 50-mL plastic centrifuge tube and the conductivity of the aqueous solution periodically measured. The tube was capped and frozen at -20 °C. Total conductivity was measured once the tube had warmed to 20 °C with shaking. The percent ion leakage was calculated as the percent of total, and subjected to an analysis to partition rates of leakage into symplastic and apoplastic components. The symplastic component was not affected by the heat-shock treatment, while the apoplastic component showed reductions consistent with reduced chilling-induced damage to the cellular membrane. The protective heat-shock treatments also significantly increased the tissues resistance to fungal infection.

A detachable porous vaginal mold facilitates reconstruction of a modified McIndoe neovagina

Objective To release a new design for a detachable porous vaginal mold to facilitate reconstruction of a modified McIndoe vaginal mold. Design We constructed a detachable, porous vaginal mold with a plastic laboratory centrifuge tube with multiple holes throughout the entire tube. Setting Patients in a national tertiary medical center. Patient(s) Four patients of congenital vaginal agenesis received the modified McIndoe procedure. Intervention(s) Two full-thickness skin grafts removed from the inguinal region were used to cover the detachable porous plastic vaginal mold; the mold was then inserted into the neovaginal cavity and kept in place by sutures between the mold and labia majora. The vaginal mold was removed on day 12 after the operation. All patients adhered to the follow-up instructions. Main outcome measure(s) Description of the accessibility of a neovaginal mold. Result(s) The vaginal mold is helpful in taking care of the vaginal wound in that it allows easy removal of wound secretion and local cleansing douches. All grafts took completely and recovered well. No detachment of the graft occurred. Conclusion(s) The advantages of the detachable porous vaginal mold made from a plastic centrifuge tube are that it is readily available, allows for easy wound care, and, as a fixed point, prevents graft detachment or inversion and thus may lead to a promising result for a modified McIndoe vaginal reconstruction.

Morphological changes of the thymus under stress caused by water immersion and restraint in SAMP1 mice

We compared the time course of the stress-induced morphological changes in the thymus between SAMP1 and C3H/He mice. The mice were kept under restraint in a 50-ml plastic centrifuge tube in water at 24° for 4 h. This treatment induced atrophy of the thymus and reduced the thymus weight, although it did not affect the body weight or major organ weights in either strains of mice. Histological studies of the thymus revealed that the stress groups of both strains showed a transient increase in the number of pyknotic cells and macrophages as compared with each control group just after the stressor treatment. Only a slight increase in the macrophage number was found at 4 h after the treatment. Furthermore, we investigated the morphological changes of the thymus until 7 days after the treatment, in order to compare the time course of recovery of the thymus atrophy between the two mouse strains. The stress groups of both strains had decreased lymphocyte populations in the thymus. The thymic lymphocyte population in C3H/He mice started to recover at 24 h after the stressor treatment. In clear contrast, the lymphocyte population in the SAMP1 thymus did not recover but continued to decrease until 7 days after the treatment, being progressively replaced by fibroblasts. These results suggest that the regenerative mechanisms of the SAMP1 thymus have some defects, and that the T-cell compartment of the immune system in SAMP1 mice may be as sensitive as aged mice from the control strain.

Use of anion-exchange membrane extraction for the high-performance liquid chromatographic analysis of mustard seed glucosinolates.

A new one-step extraction using anion-exchange membranes for the HPLC determination of glucosinolates in mustard seeds is reported. The exchange of glucosinolates on the membranes was studied using sinigrin in solutions and sinigrin added as an internal standard to seeds of yellow mustard. By varying time of extraction, membrane size, and sample size, the optimal conditions for maximum glucosinolate recovery were determined and the following procedure was adopted: 0.2 g of ground mustard seeds are heated in 20 mL of boiling water for 5 min. After cooling, samples are transferred to plastic centrifuge tubes, 9-cm(2) membranes are added, and suspensions are shaken on a mechanical shaker for 2.5 h. Glucosinolates are then eluted from the membranes with 25 mL of 1 N KCl by shaking again for 2.5 h. Using this procedure, the sinigrin extraction from solutions and from mustard seeds was linear with 80% recovery. Seeds of yellow, brown, oriental, and Indian mustard were analyzed by this procedure; excellent reproducibility, with coefficients of variation in the range 1.0-4.3% were obtained. This method offers a simple and inexpensive alternative to complicated and tedious procedures for glucosinolate isolation/purification required for chromatographic determinations.

Structural and Functional Characterization of Platelet Receptor-mediated Factor VIII Binding

Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.