Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

Background  

Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.     

Effects of supplemental vitamin E during the mature period on the reproduction performance of Taiwan Native Chicken cockerels

One-day-old Taiwan native male chicks were fed with maize-soybean rearing diets without supplemental vitamin E to 23 weeks of age. From 23 to 52 weeks of age, the cockerels (n = 90) were assigned at random to 5 dietary treatments and fed with maize-soybean diets supplemented with 0, 20, 40, 80 and 160 mg/kg of vitamin E (dl-alpha-tocopherol acetate). Pullets (225) of the same age were fed with standard diets throughout. They were artificially inseminated with one dose of 0.04 ml/bird intact and 5-fold diluted pooled semen at 31 to 43 weeks of age and at 49 weeks of age, respectively. The criteria evaluated included: semen quality, fertility and maximum and effective duration of fertility, blood characteristics, body and testes weight. Supplemental vitamin E did not affect cockerels' effective duration of fertility and percentage of fertility. However, when pullets were inseminated with diluted semen, supplementing 160 mg/kg vitamin E increased the maximum duration of fertility at 49 weeks of age. Cockerels receiving 40 to 160mg/kg supplements had higher sperm viability and motility after 39 weeks of age and those fed 80 mg/kg had higher sperm concentration at 39 weeks of age. Cockerels receiving supplements of more than 40 mg/kg vitamin E had higher body weight gain. Plasma cholesterol and testosterone were not affected by supplemental vitamin E. However, plasma luteinising hormone (LH) concentration was lower in cockerels fed 160 mg/kg. Lack of supplemental vitamin E over 39 weeks was associated with lower semen quality but did not reduce the proportion of fertile eggs laid by inseminated hens, perhaps because the insemination dose compensated for low sperm quality. We found that the maximum duration of fertility might be improved by supplementing 160 mg/kg vitamin E at 49 weeks of age.     

Somatotropin and adipose tissue metabolism: substrate and temporal effects

The purpose of these studies was to determine the time course for changes in feed intake, blood metabolites, and lipogenic activity in adipose tissue in response to the initiation of porcine somatotropin (pST) treatment and following withdrawal from treatment in barrows. An initial study was conducted to determine the impact of chronic pST treatment (4 wk of daily injection; 0 vs 4 mg/d) on adipose tissue lipid metabolism in barrows (initial weight 67 kg). Feed efficiency was improved 27%, backfat thickness was decreased 43%, and glucose and lactate oxidation and incorporation into lipid in adipose tissue was reduced 70 to 86% in pST-treated pigs. Palmitate esterification was decreased 44%, whereas palmitate oxidation was unaffected. In vitro metabolism of lactate, glucose, and palmitate in liver slices was not affected by pST treatment. The time-course for changes in intake and adipose tissue metabolism in response to 7 d of pST (0 vs 4 mg/d) treatment and 7 d of withdrawal was examined in subsequent studies in barrows (initial weight 75 kg). Feed intake during pST treatment was significantly (P < .05) less than in control pigs within 24 h of the initiation of treatment and remained low through 3 d after withdrawal. Adipose tissue biopsies were obtained on d 0, 1, 2, 4, and 7 of the treatment phase and on d 2, 4, and 7 after withdrawal from 7 d of treatment. Maximal inhibition of lipogenesis by pST treatment in adipose tissue in vitro was observed on d 4 (-68%) and d 7 (-69%). Similarly, fatty acid synthase activity declined during the treatment period, with the greatest change noted on d 7 (-26%). After withdrawal from treatment, lipogenesis gradually increased, returning to control values 7 d after withdrawal. Levels of IGF-I began to increase from d 1 to d 7 of treatment, continually decreased during withdrawal, and were normalized by the end of the withdrawal period. Plasma urea nitrogen concentrations decreased during treatment, increased during the withdrawal phase, and were normalized 4 d after the last pST treatment. Overall results indicate that most of the metabolic changes in response to pST occur within 1 wk of treatment and return to pretreatment values after 7 d of withdrawal from treatment.     

Variation in growth rates and aggressiveness of naturally occurring self‐fertile and self‐sterile isolates of the wilt pathogen Ceratocystis albifundus

Ceratocystis albifundus is the most important fungal pathogen of black wattle (Acacia mearnsii) grown in plantations in southern and eastern Africa. It is a homothallic fungus but also undergoes unidirectional mating type switching. As a result, the ascospore progeny can be either self‐fertile or self‐sterile. The only apparent difference between these mating types is the deletion of the MAT1‐2‐1 gene in self‐sterile isolates. There is some evidence suggesting that self‐sterile isolates grow more slowly than self‐fertile isolates, but this has not been tested rigorously. The aim of this study was to determine whether self‐sterile isolates are less fit by examining growth rate, relative germination rate and pathogenicity. Five self‐sterile isolates were generated from each of five self‐fertile isolates of C. albifundus and these 30 isolates were compared. The results showed that the self‐sterile isolates grew consistently slower and were less pathogenic than the self‐fertile isolates. The germination ratio of self‐fertile to self‐sterile isolates from single ascospores collected from the ascomata of five self‐fertile isolates was on average 7:3. This could be a consequence of the self‐sterile isolates having a lower germination rate. This observation, and the lower growth and pathogenicity levels, suggests that self‐sterile isolates are not likely to compete effectively in nature, raising intriguing questions regarding their role and value to C. albifundus and other fungi having a similar mating system.     

A multi-method approach to delineate and validate migratory corridors

Context

Managers are faced with numerous methods for delineating wildlife movement corridors, and often must make decisions with limited data. Delineated corridors should be robust to different data and models.

Objectives

We present a multi-method approach for delineating and validating wildlife corridors using multiple data sources, which can be used conserve landscape connectivity. We used this approach to delineate and validate migration corridors for wildebeest (Connochaetes taurinus) in the Tarangire Ecosystem of northern Tanzania.

Methods

We used two types of locational data (distance sampling detections and GPS collar locations), and three modeling methods (negative binomial regression, logistic regression, and Maxent), to generate resource selection functions (RSFs) and define resistance surfaces. We compared two corridor detection algorithms (cost-distance and circuit theory), to delineate corridors. We validated corridors by comparing random and wildebeest locations that fell within corridors, and cross-validated by data type.

Results

Both data types produced similar RSFs. Wildebeest consistently selected migration habitat in flatter terrain farther from human settlements. Validation indicated three of the combinations of data type, modeling, and corridor detection algorithms (detection data with Maxent modeling, GPS collar data with logistic regression modeling, and GPS collar data with Maxent modeling, all using cost-distance) far outperformed the other seven. We merged the predictive corridors from these three data-method combinations to reveal habitat with highest probability of use.

Conclusions

The use of multiple methods ensures that planning is able to prioritize conservation of migration corridors based on all available information.

     

Effects of Vibrio harveyi on plasma clotting protein (coagulogen) of white shrimp Litopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca²⁺) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca²⁺ ion.     

Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

A preliminary study of dietary protein requirement of juvenile marbled flounder (Pseudopleuronectes yokohamae)

An 8-week feeding trial was conducted to determine the optimal dietary protein level for juvenile marbled flounder. Five semi-purified test diets were formulated to contain different protein levels (CP) including 42.7%, 47.4%, 53.3%, 58.8%, and 64.5% (dry matter), named as CP42.7, CP47.4, CP53.3, CP58.8, and CP64.5, respectively. Five hundred and twenty-five juveniles (6.0 ± 0.1 g) were randomly distributed into 15 tanks (300 L tanks), resulting in 35 fish per tank (n = 3 tanks). Fish were fed the test diets 5 times per day until satiation. The CP58.8 resulted in the highest gain in weight and the best efficiency in feed utilization among the tested protein levels (P < 0.05). Fish fed the CP58.8 diet showed significantly higher whole-body protein and lipid contents than the fish that were fed the other diets (P < 0.05). Fish fed the CP53.3, CP58.8, and CP64.5 diets showed a significantly higher dorsal-muscle lipid content than the fish that were fed the CP42.7 and CP47.4 diets (P < 0.05). The one-slope straight broken-line regression analysis on the results of the thermal growth coefficient and feed conversion ratio indicated that the estimated optimum dietary protein level was 58.8%. Taken together, it is suggested that the dietary protein level of 58.8% is optimal for better growth and high efficiency in feed utilization for the juvenile marbled flounder.     

A Greenhouse Test for Screening Sugar Beet (Beta Vulgaris) for Resistance to Rhizoctonia Solani

Rhizoctonia solani Kühn is a serious plant pathogenic fungus, causing various types of damage to sugar beet (Beta vulgaris L.). In Europe, the disease is spreading and becoming a threat for the growing of this crop. Plant resistance seems to be the most practical and economical way to control the disease. Experiments were carried out to optimise a greenhouse procedure to screen plants of sugar beet for resistance to R. solani. In the first experiment, two susceptible accessions were evaluated for root and leaf symptoms, after being grown in seven different soil mixtures and inoculated with R. solani. The fungus infected all plants. It was concluded that leaf symptoms were not reliable for the rating of disease severity. Statistically significant differences between the soil mixtures were observed, and there were no significant differences between the two accessions. The two soil mixtures, showing the most severe disease symptoms, were selected for a second experiment, including both resistant and susceptible accessions. As in the first experiment, root symptoms were recorded using a 1–7 scale, and a significant expression of resistance was observed. The average severity of the disease in the greenhouse experiment generally was comparable with the infection in field experiments, and the ranking of the accessions was the same in the two types of experiments. It was concluded that evaluation procedures in the greenhouse could be used as a rapid assay to screen sugar beet plants for resistance to R. solani.     

Comparison of arylalkylamine N-acetyltransferase and melatonin receptor type 1B immunoreactivity between young adult and aged canine spinal cord

Melatonin affects diverse physiological functions through its receptor and plays an important role in the central nervous system. In the present study, we compared immunoreactivity patterns of arylalkylamine N-acetyltransferase (AANAT), an enzyme essential for melatonin synthesis, and melatonin receptor type 1B (MT2) in the spinal cord of young adult (2~3 years) and aged (10~12 years) beagle dogs using immunohistochemistry and Western blotting. AANAT-specific immunoreactivity was observed in the nuclei of spinal neurons, and was significantly increased in aged dog spinal neurons compared to young adult spinal neurons. MT2-specific immunoreactivity was found in the cytoplasm of spinal neurons, and was predominantly increased in the margin of the neuron cytoplasm in aged spinal cord compared to that in the young adult dogs. These increased levels of AANAT and MT2 immunoreactivity in aged spinal cord might be a feature of normal aging and associated with a feedback mechanism that compensates for decreased production of melatonin during aging.