Microtiter micromass cultures of limb-bud mesenchymal cells

Summary A method is described for growing high-density micromass cultures of chick and mouse limb mesenchyme cells in 96-well microtiter plates (μTμM cultures). Rapid quantitative estimates of chondrogenic expression were obtained by automated spectrophotometric analysis of Alcian-blue-stained cartilage matrix extracts performed in the wells in which the cells had been grown. Quantitative estimates of myogenic expression were obtained similarly using anti-sarcomere myosin monoclonal antibody and modified ELISA techniques. This μTμM-ELISA method may be adapted for use with other antigens for which specific antibodies are available. These methods were used to compare cartilage and muscle differentiation in 1 to 4 d μTμM cultures grown in serum-containing (SCM) and defined (DM) media. The DM contains minimal additives (insulin, hydrocortisone, and in some cases, ascorbate or transferrin) and supports both chondrogenesis and myogenesis. The colorimetric analyses agree well with the morphologic appraisal of chondrogenesis and myogenesis. Similar numbers of cartilage nodules formed in all cultures, but in DM the nodules failed to enlarge; explaining the reduced matrix synthesis in DM as compared with SCM, and suggesting that nodule enlargement is a discrete, serum-dependent step. Studies of selected additives to DM show that transferrin enhances myogenesis, ascorbic acid enhances chondrogenesis, and retinoic acid inhibits chondrogenesis. Together, the μTμM system, in situ colorimetric assays of chondrogenesis and myogenesis, and DM will allow rapid prescreening of teratogens and screening of various bioactive compounds (e.g., hormones, growth factors, vitamins, adhesion factors) for effects on limb mesenchymal cell differentiation. This work was supported by grants RR08006-13 (DFP) and HD05505 and HD18577 (MS) from the National Institutes of Health, Bethesda, MD. MF-20 hybridoma supernatant was obtained from the Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, Iowa 52242 (maintained by NIH grant NO1-HD62915).     

检测特异性抗体分泌细胞(ASCs)的固相酶联免疫斑(ELISPOT)技术的建立

ＥＬＩＳＰＯＴ技术是目前国外评价疫苗免疫原性和免疫应答机理研究中最常使用的方法。本文用自制的ＮＣ膜２４孔板为固相支持物,建立了检测小鼠脾细胞中特异性ＡＳＣｓ的ＥＬＩＳＰＯＴ技术,在检测方法的特异性、结果的稳定性上均取得了满意的效果。并对该方法的某些实验条件进行了摸索。     

丙型肝炎病毒实验诊断技术的研究进展

目前,世界各国医学界均在积极探索非甲非乙型肝炎病毒的病原。随着分子克隆技术及特异性丙肝病毒抗原的出现,使特异性丙肝检测手段迅速发展,各种类型的诊断试剂盒不断推出,并在敏感性和待异性方面均有明显提高,日趋发展为简便、快速、可靠的方法。本文特就有关丙肝分子生物学及实验诊断技术的研究进展作一综述。     

Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions

A highly active inhibitor of the binding of estrone and estradiol-17β to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH₂₀ and thin layer chromatography : it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17β anti-bodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.     

Stooping,crouching, and standing – Characterizing balance control strategies across postures

BackgroundWhile stooping and crouching postures are critical for many activities of daily living, little is known about the balance control mechanisms employed during these postures. Accordingly, the purpose of this study was to characterize the mechanisms driving net center of pressure (COP_Net) movement across three postures (standing, stooping, and crouching) and to investigate if control in each posture was influenced by time.MethodsTen young adults performed the three postures for 60 s each. Kinetic signals were collected via a force platform under each foot. To quantify mechanisms of control, correlations (Correl_LR) were calculated between the left and right COP trajectories in the anterior-posterior (AP) and medio-lateral (ML) directions. To examine the potential effects of time on balance control strategies, outcomes during the first 30 s were compared to the last 30 s.ResultsCorrel_LR values did not differ across postures (AP: p = 0.395; ML: p = 0.647). Further, there were no main effects of time on Correl_LR (AP: p = 0.976; ML: p = 0.105). A significant posture-time interaction was observed in the ML direction (p = 0.045) characterized by 35% decreases in Correl_LR over time for stooping (p = 0.022).ConclusionThe dominant controllers of sway (i.e., AP: ankle plantar/dorsi flexors; ML: hip load/unload mechanism) are similar across quiet stance stooping, and crouching. Changes in ML control strategies over time suggests that fatigue could affect prolonged stooping more so than crouching or standing.     

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD_260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.     

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade‐off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier–Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.     

Regeneration of specific innervation in Xenopus pectoralis muscle

We investigated the motor unit organization and precision of reinnervation in the Xenopus pectoralis muscle following different manipulations, including crush or section of the posterior pectoralis nerve, foreign nerve innervation, and crush coupled with activity modulation or block. Most fibers have two neuromuscular junctions, and multielectrode recordings were used to identify the axonal origin of all inputs to both junctions on most or all fibers covering about 25% of the muscle surface. Following simple nerve crush, a highly organized innervation pattern was restored, indistinguishable from the normal pattern, including selective innervation of fibers of similar input resistance (R_in), compact motor unit organization, and high incidence of exclusive innervation of both end plates on each fiber by the same axon (distributed mononeuronal innervation, or a/a pattern). Initial reinnervation was equally precise when nerve conduction in the regenerating nerve was blocked by tetrodotoxin. More distant or repeated nerve crush or nerve section delayed and reduced the precision of reinnervation, but the majority of fibers still received input to both end plates by the same axon, often in combination with others. A foreign nerve, the pectoralis sternalis, which in its own muscle forms only single end plates, showed less precise reinnervation, but still had an incidence of a/a innervation far above chance. These data imply the expression and recognition of remarkably precise chemospecific cues even in mature animals, superimposed on which is a further refinement by synapse elimination, probably based on an activity-dependent process. © 1996 John Wiley & Sons, Inc.     

Morphology and development of additional bony elements in the genus Brachycephalus (Anura: Brachycephalidae)

We describe the extra bony elements, plates, and osteoderms present in species of the genus Brachycephalus. Samples of eight species of Brachycephalus, including seven populations of Brachycephalus ephippium, were examined. The large additional elements associated with the skull (parotic plate) and vertebrae (vertebral and paravertebral plates) all comprise intramembranous bone, similar to that of the frontoparietal or nasal bones of the skull of most of frogs. Additionally, in the dermis of one unnamed species, we discovered and described true osteoderms. We discuss the morphological nature and diversity of theses elements and their importance as evidence of phylogenetic relationship within Brachycephalus. In summary, three distinct conditions of extra bony elements occur in the genus Brachycephalus: (1) bony plates may be present or absent in species of the genus; (2) a few, small bony plates may be developed and these may be represented by (a) paravertebral plates small and restricted to the distal ends of the transverse processes of the presacral IV, (b) parotic plates small and not covering the tops of the squamosals, and (c) ornamented spinal plates on all vertebrae; and (3) well‐developed bony plates may be present as (a) paravertebral plates forming a ‘bone‐shield’ on the dorsal surface of the trunk, ornamented, and visible through the integument, (b) parotic plates covering the tops of the squamosals, and (c) spinal plates associated with all vertebrae, and ornamented on vertebrate I–VI. Although the phenomenon of miniaturization may be associated with the appearance of new elements in at least some of the species in the genus, the traditional rule may not be universally applicable. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 752–767.     

On the origin of the putative furca of the Ostracoda (Crustacea)

The posterior end of body of the extant ostracods exhibits a pair of variously shaped appendages, commonly designated as furca(e), uropods or caudal rami, used for feeding and/or locomotion. It is here shown that the so-called furca of all extant ostracods has evolved from the (probably epipodal) vibratory plates of a pair of uropods. The transformation comprised the following steps: (a) complete reduction of the uropodal protopodite and endopodite; (b) sclerotisation of the lateral walls of the vibratory plates; (c) transformation of the branchial filaments into spines and/or claws; (d) re-orientation of the plates from posterodorsal to posteroventral. These modifications are suggested to have evolved in parallel with a change in function, from respiratory to locomotory and/or feeding. The most primitive condition, reminiscent of the ancestral state of character, is seen in the Platycopida: the ‘furca’ still appears similar in shape to the vibratory plates of the pair of sixth limbs. In the Podocopida the uropodal plates have been modified into plate-like, more often into rod-shaped rami mainly used for locomotion. In both the Platycopida and Podocopida the anus has remained in its original place, posterior to the ‘furcal’ plates or rami. In the Myodocopida and Halocyprida the uropodal vibratory plates are transformed into heavily developed lamellae bearing sturdy spines. They are activated by a complex apparatus of muscles and sclerites, the development of which necessitated the displacement of the anus from the end of the body towards its present place, anterior to the ‘furca’. The furca of the Ostracoda being not a ‘true’ furca, a change in terminology is proposed: uropodal plates or lamellae in the Platycopida, Palaeocopida and Myodocopida/Halocyprida; uropodal rami in the Podocopida. The so-called furcae of the Ostracoda being homologous structures, it is concluded that all extant ostracods belong to a monophyletic lineage.