Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

The autophagy gene ATG5 plays an essential role in B lymphocyte development

Macroautophagy (herein autophagy) is an evolutionarily conserved process, requiring the gene ATG5, by which cells degrade cytoplasmic constituents and organelles. Here we show that ATG5 is required for efficient B cell development and for the maintenance of B-1a B cell numbers. Deletion of ATG5 in B lymphocytes using Cre-LoxP technology or repopulation of irradiated mice with ATG5-/- fetal liver progenitors resulted in a dramatic reduction in B-1 B cells in the peritoneum. ATG5-/- progenitors exhibited a significant defect in B cell development at the pro- to pre-B cell transition, although a proportion of pre-B cells survived to populate the periphery. Inefficient B cell development in the bone marrow was associated with increased cell death, indicating that ATG5 is important for B cell survival during development. In addition, B-1a B cells require ATG5 for their maintenance in the periphery. We conclude that ATG5 is differentially required at discrete stages of development in distinct, but closely related, cell lineages.     

地中海沿岸沙丘土壤种子库的异质性

应用自然萌发法和物理筛选法相结合的方法,调查了地中海沿岸沙丘三种微生境土壤总种子库和永久种子库特征,用功能群的方法分析了微生境之间的土壤总种子库和永久土壤种子库的组成和结构差异,也分析了主要物种土壤种子库的微生境分布格局,分析了种子大小和土壤种子库中的种子永久性关系。发现地中海沿岸沙丘土壤种子库具有明显的空间异质性,即使在相同的微生境内,土壤种子库的分布也不是均匀的,甚至具有很大的差异;微生境对总的土壤种子库和各功能群的土壤种子库的分布格局具有显著的影响,开阔地区域具有最大的种子密度,而灌丛下和路径具有较小的种子密度;在永久土壤种子库中,豆科类植物种子在休眠种子中占有最大的比例,约为76％,而一年生和多年生禾草未发现休眠种子,豆类、伞形科类、多年生阔草、菊科类、一年生阔叶草和十字花科类等种子在土壤中的永久性比例依次下降,分别为50％、46％、41％、6％、6％和1％,微生境影响各功能群种子在土壤中的永久性。微生境显著影响大部分主要物种的土壤种子库分布;种子大小与其在土壤中的永久性为正相关,即大种子具有较高的永久性,小种子具有较低的永久性。     

Quantum mechanical origin of the conformational preferences of 4-thiaproline and its <Emphasis Type="Italic">S</Emphasis>-oxides

The saturated ring and secondary amine of proline spawn equilibria between pyrrolidine ring puckers as well as peptide bond isomers. These conformational equilibria can be modulated by alterations to the chemical architecture of proline. For example, C^γ in the pyrrolidine ring can be replaced with sulfur, which can be oxidized either stereoselectively to yield diastereomeric S-oxides or completely to yield a sulfone. Here, the thiazolidine ring and peptide bond conformations of 4-thiaproline and its S-oxides were analyzed in an Ac–Xaa–OMe system using NMR spectroscopy, X-ray crystallography, and hybrid density functional theory. The results indicate that the ring pucker of the S-oxides is governed by the gauche effect, and the prolyl peptide bond conformation is determined by the strength of the n → π* interaction between the amide oxygen and the ester carbonyl group. These findings, which are consistent with those of isologous 4-hydroxyprolines and 4-fluoroprolines, substantiate the importance of electron delocalization in amino acid conformation.     

Direct utilization of mannose for mammalian glycoprotein biosynthesis

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2- 3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic- derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.      

Evaluation of the Wii Balance Board for Walking Aids Prediction: Proof-of-Concept Study in Total Knee Arthroplasty

Background and Objectives

To provide proof-of-concept for the validity of the Wii Balance Board (WBB) measures to predict the type of walking aids required by inpatients with a recent (≤4days) total knee arthroplasty (TKA).

Methods

A cross-sectional sample of 89 inpatients (mean age, 67.0±8years) with TKA was analyzed. A multivariable proportional odds prediction model was constructed using 8 pre-specified predictors – namely, age, sex, body mass index, knee pain, knee range-of-motion, active knee lag, and WBB-derived standing balance. The type of walking aids prescribed on day 4 post-surgery was the outcome of interest – an ordinal variable with 4 categories (walking stick, narrow- and broad-base quadstick, and walking frame).

Results

Women, increasing body mass index, and poorer standing balance were independently associated with greater odds for requiring walking aids with a larger base-of-support. The concordance-index of the prediction model was 0.74. The model comprising only WBB-derived standing balance had nearly half (44%) the explanatory power of the full model. Adding WBB-derived standing balance to conventional demographic and knee variables resulted in a continuous net reclassification index of 0.60 (95%CI,0.19-1.01), predominantly due to better identification of patients who required walking aids with a large base-of-support (sensitivity gain).

Conclusions

The WBB was able to provide quantitative measures of standing balance which could assist healthcare professionals in prescribing the appropriate type of walking aids for patients. Further investigation is needed to assess whether using the WBB could lead to meaningful changes in clinical outcomes such as falls.     

Pericyte response during choriocapillaris atrophy in the rabbit

The rabbit and rat choriocapillaris atrophies in response to experimental destruction of the retinal pigment epithelium by intravenous injection of sodium iodate. This provides a convenient model of capillary atrophy. We have observed that pericytes are spared during this process; the atrophy is due to loss of endothelium only. Extensive examination of thin sections obtained 1 day to 11 weeks after administration of iodate showed that pericytes retained their normal relationship to the remnant capillary basement membrane left behind as the endothelial tube atrophied. This was most conspicuously manifested in their retention of processes longitudinally disposed along the sleeves of remnant basement membrane. The processes retained bundles of actin filaments that had dense regions along them and inserted into subplasmalemmal densities at basement membrane attachment sites, i.e. they had the characteristics of stress fibers. The pericytes did not phagocytose the debris of endothelial necrosis, in spite of their known phagocytic abilities. Necrotic endothelial cells were eliminated by sloughing into the capillary lumen. The observations support the idea that the function of pericytes in the choriocapillaris, the major source of nutrition for the retinal photoreceptors, resides in their contractility, and that pericytes do not remove necrotic endothelium during capillary atrophy.     

Retention of shoot regeneration capacity of tobacco callus by Na2SO4

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown in the light on shoot-forming medium in the presence of Na₂SO₄ for over a year. An increase in Na₂SO₄ concentration resulted in decreasing callus growth, decreasing percentage of calli producing shoots and number of shoots per callus, and increasing callus percent dry weight. Regeneration of shoots from callus grown in the absence of Na₂SO₄ began to decline after 14 months in culture, and shoot regeneration capacity was completely lost after 18 months. In contrast, 18-month old callus continuously grown in the presence of Na₂SO₄ retained the ability to form shoots. The highest percent of callus pieces that formed shoots and the maximum number of shoots per callus occurred at 70 mM (1%) Na₂SO₄. All plants arising from the 18-month old callus were polyploid. Both 9- and 18-month old callus exhibited more negative water and osmotic potentials in the presence of increasing Na₂SO₄ concentration.Abbreviations water potential - _S osmotic potential - CGI callus growth index     

Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations

An accurate, precise, and convenient procedure was developed for measurement of the latency of the low-Km mannose-6-phosphatase activity for the purpose of assessment of the membrane permeability barrier in microsomes. This approach is based on previous work of Arion et al. [J. Biol. Chem. (1976) 251, 4901-4907] and consists of measurement of mannose-6-phosphatase activity in the untreated microsomal fraction and in the corresponding microsomes that are fully disrupted in order to eliminate the membrane permeability barrier. Complete disruption of rat liver microsomes was achieved by incubation for 60 min at 0 degree C in the presence of 4 mM zwitterionic detergent 3-[(3-cholamido-propyl)dimethyl-ammonio]-2-hydroxy-1-propane sulphonate (Chapso). That the microsomal membrane permeability barrier was eliminated under those conditions was suggested by the fact that the enzyme activation (up to 50-fold) produced by this pretreatment was at least as large as the effect of any other previously reported disruptive procedure. Disruption of the microsomes by Chapso or by ultrasonication markedly enhanced the thermolability of the mannose-6-phosphatase activity. In addition, exposure of the microsomes to high concentrations of Chapso produced enzyme inactivation that could be partially reversed by dilution of the detergent prior to assaying the enzymic activity. Investigation of these enzyme inactivation phenomena under various incubation conditions for disruption of the microsomes by Chapso and for subsequent assay of mannose-6-phosphatase activity in the presence of Chapso enabled us to define conditions under which instability of the enzyme was undetectable. Using these optimized procedures for disruption of microsomes and assay of hexose-6-P phosphohydrolase, we found that the low-Km mannose-6-phosphatase activity of untreated rat liver microsomes consistently was less than 5% of the total enzyme activity in the fully disrupted microsomes. Accurate and precise assay of the structural latency of mannose-6-phosphatase in membrane preparations must be performed under well-controlled conditions, with special attention to the marked thermolability of the enzyme in the presence of detergent, and is a prerequisite for using this approach for the purpose of assessing intactness of microsomal preparations.     

Some morphogenic effects of sodium sulfate on tobacco callus

Callus cultures of Nicotiana tabacum L cv. Wisconsin 38 were initiated and grown on shoot-forming (SF) and callus proliferation (CP) medium with or without Na₂SO₄. Two cultures were maintained on SF medium with 0, 0.75, 1 or 1.5% Na₂SO₄ for 2.5 and 3.5 years. In the older culture only callus grown on salt formed shoots throughout the maintenance period, while in the younger culture the control responded best and Na₂SO₄ was inhibitory. Callus from the older culture which had been grown on salt continued to form shoots in the absence of salt. Na₂SO₄ caused adventitious shoot formation in three cultures on CP medium. These shoots were present for 7 subcultures after removal of Na₂SO₄; but established, control callus, did not form shoots when transferred to Na₂SO₄. Callus initiated and maintained on NaCl or mannitol showed a slight increase in shoot initiation. On NaCl, Na₂SO₄ or mannitol, the tissue osmotic potential became more negative and proline concentration increased.