Reversible dissociation of the plant golgi apparatus by brefeldin A

Summary— The effects of the drug Brefeldin A, shown to block the translocation of proteins between the endoplasmic reticulum and the Golgi apparatus in animal cells, were studied on different plant cell systems. In suspension culture cells and root tissues, the Golgi aparatus was affected by Brefeldin A treatments resulting in distortion and dissociation of the Golgi stacks, coupled with appearance of numerous vesicles in the cytoplasm. This process was reversible. Therefore, Brefeldin A provides a powerful tool with which to study Golgi dynamics and function in plant as well as in animal cells.     

Computer simulations improve university instructional laboratories

Laboratory classes are commonplace and essential in biology departments but can sometimes be cumbersome, unreliable, and a drain on time and resources. As university intakes increase, pressure on budgets and staff time can often lead to reduction in practical class provision. Frequently, the ability to use laboratory equipment, mix solutions, and manipulate test animals are essential learning outcomes, and "wet" laboratory classes are thus appropriate. In others, however, interpretation and manipulation of the data are the primary learning outcomes, and here, computer-based simulations can provide a cheaper, easier, and less time- and labor-intensive alternative. We report the evaluation of two computer-based simulations of practical exercises: the first in chromosome analysis, the second in bioinformatics. Simulations can provide significant time savings to students (by a factor of four in our first case study) without affecting learning, as measured by performance in assessment. Moreover, under certain circumstances, performance can be improved by the use of simulations (by 7% in our second case study). We concluded that the introduction of these simulations can significantly enhance student learning where consideration of the learning outcomes indicates that it might be appropriate. In addition, they can offer significant benefits to teaching staff.     

The crystal structure of a high affinity RNA stem-loop complexed with the bacteriophage MS2 capsid: further challenges in the modeling of ligand-RNA interactions

We have determined the structure to 2.8 A of an RNA aptamer (F5), containing 2'-deoxy-2-aminopurine (2AP) at the -10 position, complexed with MS2 coat protein by soaking the RNA into precrystallised MS2 capsids. The -10 position of the RNA is an important determinant of binding affinity for coat protein. Adenine at this position in other RNA stem-loops makes three hydrogen bonds to protein functional groups. Substituting 2AP for the -10 adenine in the F5 aptamer yields an RNA with the highest yet reported affinity for coat protein. The refined X-ray structure shows that the 2AP base makes an additional hydrogen bond to the protein compared to adenine that is presumably the principal origin of the increased affinity. There are also slight changes in phosphate backbone positions compared to unmodified F5 that probably also contribute to affinity. Such phosphate movements are common in structures of RNAs bound to the MS2 T = 3 protein shell and highlight problems for de novo design of RNA binding ligands.     

Indirect competitive immunoassay for detection of aflatoxin B1 in corn and nut products using the array biosensor

Because of the potential health risks of aflatoxin B₁ (AFB₁), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB₁ in buffer, corn and nut products. AFB₁-spiked foods were extracted with methanol and Cy5-anti-AFB₁ added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB₁. The resulting fluorescence signal decreased as the concentration of AFB₁ in the sample increased. The limit of detection for AFB₁ in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.     

Development of Health‐Related Waist Circumference Thresholds Within BMI Categories

Objective: To develop and cross‐validate waist circumference (WC) thresholds within BMI categories. The utility of the derived values was compared with the single WC thresholds (women, 88 cm; men, 102 cm) recommended by NIH and Health Canada. Research Methods and Procedures: The sample included adults classified as normal weight (BMI = 18.5 to 24.9), overweight (BMI = 25 to 29.9), obese I (BMI = 30 to 34.9), and obese II+ (BMI ≥ 35) from the Third U.S. National Health and Nutrition Examination Survey (NHANES III; n = 11, 968) and the Canadian Heart Health Surveys (CHHS; n = 6286). Receiver operating characteristic curves were used to determine the optimal WC thresholds that predicted high risk of coronary events (top quintile of Framingham scores) within BMI categories using the NHANES III. The BMI‐specific WC thresholds were cross‐validated using the CHHS. Results: The optimal WC thresholds increased across BMI categories from 87 to 124 cm in men and from 79 to 115 cm in women. The validation study indicated improved sensitivity and specificity with the BMI‐specific WC thresholds compared with the single thresholds. Discussion: Compared with the recommended WC thresholds, the BMI‐specific values improved the identification of health risk. In normal weight, overweight, obese I, and obese II+ patients, WC cut‐offs of 90, 100, 110, and 125 cm in men and 80, 90, 105, and 115 cm in women, respectively, can be used to identify those at increased risk.     

Ovine Model for Critical-Size Tibial Segmental Defects

A segmental tibial defect model in a large animal can provide a basis for testing materials and techniques for use in nonunions and severe trauma. This study reports the rationale behind establishing such a model and its design and conclusions. After ethics approval of the study, aged ewes (older than 5 y; n = 12) were enrolled. A 5-cm mid diaphyseal osteoperiosteal defect was made in the left tibia and was stabilized by using an 8-mm stainless-steel cross-locked intramedullary nail. Sheep were euthanized at 12 wk after surgery and evaluated by using radiography, microCT, and soft-tissue histology techniques. Radiology confirmed a lack of hard tissue callus bridging across the defect. Volumetric analysis based on microCT showed bone growth across the 16.5-cm³ defect of 1.82 ± 0.94 cm³. Histologic sections of the bridging tissues revealed callus originating from both the periosteal and endosteal surfaces, with fibrous tissue completing the bridging in all instances. Immunohistochemistry was used to evaluate the quality of the healing response. Clinical, radiographic, and histologic union was not achieved by 12 wk. This model may be effective for the investigation of surgical techniques and healing adjuncts for nonunion cases, where severe traumatic injury has led to significant bone loss.Abbreviations: BMP2, bone morphogenic protein 2; CATK, cathepsin K; VEGF, vascular endothelial growth factorThe human tibia is the most frequently broken long bone, often with significant bone loss.⁴ Segmental tibial defects can occur as a result of large tumor removal, trauma such as motor vehicle accidents, and more recently, blast injuries as seen with the escalating number of global conflicts. Treatment of these large bone and surrounding soft tissue defects is an ongoing, costly, and challenging clinical problem; no surgical technique has currently achieved preeminence.⁴ The general consensus on factors that affect healing include concomitant disease, age, and degree of trauma.⁵ When the first 2 factors, which are patient-related, are removed from the equation, healing is influenced by the size, anatomic location, and soft-tissue coverage of the defect. The ability to study these situations in a well-controlled, robust, and reproducible preclinical model would be advantageous to help establish effective surgical techniques and evaluate implants and materials.A literature review revealed that many ovine models for bone defects have been used, but all have limitations^{6,12,14,15,20,21,24,25,27,31,37,39,40} (Figure 1). Variations in protocols, such as age of the animals, size of the defect, and the bone and stabilization techniques used, limit meaningful comparison between studies.^33,34 Although some studies have investigated material performance in the healing of defects, they did not rigorously quantify control defects,^17,20 and others used no controls at all.³⁹ There is often no explanation regarding the use of a particular defect size, leading to the question of whether the defect size was critical.²⁴ The choice of bone used has been also varied; the femur,¹⁵ tibia,³⁷ and metatarsus⁴⁰ have all been studied. A noncritical-size defect implies that healing would eventually occur without the presence of any graft materials. One study,¹² for example, used a 3-cm defect at an average of 1.8 times the diameter of the tibias in question and found that empty controls achieved as much as 26% of the stiffness of an intact tibia after 12 wk. Stabilization methods include plating,^21,40 external fixtures,²⁰ intramedullary nails,^6,16 and a combination of intramedullary nails and plating.³⁷Open in a separate windowFigure 1.A limited summary of the many studies where a segmental tibial has been used with their references.The criteria used in the present study for a critical-size segmental tibial defect model were based on the following factors. The ovine tibia closely resembles that of the human tibia in terms of size, shape, and physical properties and is commonly used when studying human orthopedic diseases.^26,34 Intramedullary nailing has become the most commonly used method of tibial fracture fixation in human orthopedic surgery.^8,22 An 8-mm intramedullary nail is commonly used in the treatment of human fractures, further confirming the size similarity between the ovine and human tibiae.¹⁹The aim of this study was to establish and characterize a preclinical ovine 5-cm osteoperiosteal critical-size tibial segmental defect model in mature sheep. The endpoints included those commonly used clinically, such as radiography and microCT. Histology to investigate the degree of healing and immunohistochemistry to characterize the healing process were included to complete the evaluation process.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Altered patterns of fractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy

The genome sequence of the paleohexaploid Brassica rapa shows that fractionation is biased among the three subgenomes and that the least fractionated subgenome has approximately twice as many orthologs as its close (and relatively unduplicated) relative Arabidopsis than had either of the other two subgenomes. One evolutionary scenario is that the two subgenomes with heavy gene losses (I and II) were in the same nucleus for a longer period of time than the third subgenome (III) with the fewest gene losses. This "two-step" hypothesis is essentially the same as that proposed previously for the eudicot paleohexaploidy; however, the more recent nature of the B. rapa paleohexaploidy makes this model more testable. We found that subgenome II suffered recent small deletions within exons more frequently than subgenome I, as would be expected if the genes in subgenome I had already been near maximally fractionated before subgenome III was introduced. We observed that some sequences, before these deletions, were flanked by short direct repeats, a unique signature of intrachromosomal illegitimate recombination. We also found, through simulations, that short--single or two-gene--deletions appear to dominate the fractionation patterns in B. rapa. We conclude that the observed patterns of the triplicated regions in the Brassica genome are best explained by a two-step fractionation model. The triplication and subsequent mode of fractionation could influence the potential to generate morphological diversity--a hallmark of the Brassica genus.     

Angiogenic factors in women ten years after severe very early onset preeclampsia

Background

Women with a history of mainly severe and early onset preeclampsia have an increased risk of future cardiovascular disease. During these complicated pregnancies increased levels of anti-angiogenic factors can be found. We hypothesize that women with a history of severe very early onset preeclampsia still have increased levels of these biomarkers years after this pregnancy, resulting in increased risk for cardiovascular disease.

Methods

Twenty women with severe early onset preeclampsia before 24 weeks'' gestation, who delivered between 1993–2003 in a tertiary referral centre and twenty matched controls with uncomplicated pregnancies and healthy term infants, were addressed for participation in the study. Venous plasma samples were analyzed for basic fibroblast growth factor (bFGF), placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1), vascular endothelial growth factor (VEGF), E- and P-selectin, soluble intercellular adhesion molecule-3 (sICAM-3) and thrombomodulin by ELISA.

Results

Sixteen case subjects and 18 control subjects consented participation. The median time interval index pregnancy to study was 9.4 and 9.7 years for cases and controls, respectively. Median levels for cases-controls (p-value) were not different; bFGF: 17.43–11.11 pg/mL (0.33), sFlt-1: 102.98–101.92 pg/ml (0.84), PLGF: 3.57–4.20 pg/mL (0.38), VEGF: 64.05–45.72 pg/mL (0.73), E-selectin: 5.11–4.68 ng/mL (0.20), P-selectin: 85.35–71.69 ng/mL (0.69), sICAM-3: 0.42–0.63 ng/mL (0.41) and Thrombomodulin: 0.92–0.93 ng/mL (0.59).

Conclusion

There were no differences in angiogenic biomarkers between women with a history of severe early onset preeclampsia versus uncomplicated pregnancy almost 10 years later, suggesting that these angiogenic factors will not contribute to the early detection of women at risk for future cardiovascular disease.     

Social Reinforcement Delays in Free-Flying Honey Bees (Apis mellifera L.)

Free-flying honey bees (Apis mellifera L.) reactions were observed when presented with varying schedules of post-reinforcement delays of 0 s, 300 s, or 600 s. We measured inter-visit-interval, response length, inter-response-time, and response rate. Honey bees exposed to these post-reinforcement delay intervals exhibit one of several patterns compared to groups not encountering delays, and had longer inter-visit-intervals. We observed no group differences in inter-response time. Honey bees with higher response rates tended to not finish the experiment. The removal of the delay intervals increased response rates for those subjects that completed the trials.