Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts

The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.     

Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult

Using multiple, independent approaches to molecular species delimitation is advocated to accommodate limitations and assumptions of a single approach. Incongruence in delimitation schemes is a potential by‐product of employing multiple methods on the same data, and little attention has been paid to its reconciliation. Instead, a particular scheme is prioritized, and/or molecular delimitations are coupled with additional, independent lines of evidence that mitigate incongruence. We advocate that incongruence within a line of evidence should be accounted for before comparing across lines of evidence that can themselves be incongruent. Additionally, it is not uncommon for empiricists working in nonmodel systems to be data‐limited, generating some concern for the adequacy of available data to address the question of interest. With conservation and management decisions often hinging on the status of species, it seems prudent to understand the capabilities of approaches we use given the data we have. Here, we apply two molecular species delimitation approaches, spedeSTEM and BPP, to the Castilleja ambigua (Orobanchaceae) species complex, a relatively young plant lineage in western North America. Upon finding incongruence in our delimitation, we employed a post hoc simulation study to examine the power of these approaches to delimit species. Given the data we collected, we find that spedeSTEM lacks the power to delimit while BPP is capable, thus allowing us to address incongruence before proceeding in delimitation. We suggest post hoc simulation studies like this compliment empirical delimitation and serve as a means of exploring conflict within a line of evidence and dealing with it appropriately.     

沿海拔梯度的表型可塑性影响银胶菊叶甲的取食效率和发育(英文)

【目的】本研究旨在弄清楚生态气候因子是否影响尼泊尔银胶菊Parthenium hysterophorus盛产区中银胶菊叶甲Zygogramma bicolorata成虫后代的形态学测量参数和基础生物学。【方法】 从尼泊尔Kathmandu (24℃, 海拔1 400 m, 温带气候), Chitwan (25℃, 海拔415 m, 热带或亚热带气候)和Mahendranagar (34℃, 海拔229 m, 湿润 亚热带气候)地区采集银胶菊叶甲成虫,在最适室内条件下评估各生态气候区中其F₁代4龄幼虫和雌成虫的取食特征。我们假定在自由取食和最适非生物条件下饲养的后代将不会受到食物和气温的间接影响,不管亲本属于哪一生态气候区,后代利用食物的能力一致。【结果】然而,我们发现,尽管在最适条件下饲养,银胶菊叶甲后代的体型和取食特征与其亲本相似。体型大的亲本(来自Kathmandu地区),其后代体型也大,但是其食物利用效率比体型小的亲本(来自Chitwan 和Mahendranagar地区)所产后代小。这3个生态气候区中,与幼虫相比正在发育的雌成虫均表现出体重增加而食物利用效率降低。【结论】这些发现说明,食物和最适非生物条件的间接影响可能不影响银胶菊叶甲后代的表型可塑性,后代的体型和取食特征与亲本相似。发生的可遗传的变化可能是由于种内的遗传多样性。我们期望这些发现有助于理解气候变化背景下银胶菊叶甲成虫的表型可塑性、分布格局和取食行为。     

Cellular contribution to symmetrical forelimbs from triploid-marked "polarizing region" in the embryo of axolotl, Ambystoma mexicanum

Grafts of posterior tissue placed anterior to the limb bud in the salamander embryo exert a polarizing influence. To explain this result, the idea that the anteroposterior axis of the developing forelimb is polarized by a diffusible morphogen has been proposed. An alternative hypothesis, and the working hypothesis of the present study, is that the polarization of the developing salamander forelimb is accomplished by short-range cellular interactions resulting in intercalation rather than by the more global influence of a diffusible morphogen. One prediction of this intercalation hypothesis is that cells will be contributed to the limb from the "polarizing tissue." To test this idea, grafts of triploid marked polarizing tissue were implanted anterior to the limb bud in 82 diploid axolotl embryos at stages 32-34 of development. A total of 27 (33%) of the limbs that resulted were symmetrical and ranged in complexity from one to seven digits. Histological analysis of a subgroup of the original symmetrical limbs revealed that mesodermally derived tissues in the anterior side of these limbs (the side which formed as a duplication in response to the influence of the graft) contained high percentages of trinucleolate cells (muscle, 12.1%; connective tissue tissue, 12.5%; and cartilage, 13.4%) when compared to similar tissues in the posterior side of the same symmetrical limbs (muscle, 1.8%; connective tissue , 0.7%; and cartilage, 0.6%). When symmetrical limbs were amputated, 73% regenerated symmetrical limbs. When these regenerated limbs were again amputated, 63% formed symmetrical secondary regenerates. Histological analysis of the first generation of regenerated limbs revealed that the pattern of distribution of trinucleolate cells in each regenerate was similar to the pattern seen in the original symmetrical limb. These results indicate that there is considerable cellular contribution to the anterior side of the symmetrical forelimb from the mesoderm of grafted "polarizing tissue." This result supports the idea that short-range cellular interaction are sufficient for formation of symmetrical forelimbs in salamander embryos.     

Formulation, evaluation, and comparison of bilayered and multilayered mucoadhesive buccal devices of propranolol hydrochloride

The purpose of this research work was to establish mucoadhesive buccal devices of propranolol hydrochloride (PRH) in the forms of bilayered and multilayered tablets. The tablets were prepared using sodium carboxymethylcellulose (SCMC) and Carbopol-934 (CP) as bioadhesive polymers to impart mucoadhesion and ethyl cellulose (EC) to act as an impermeable backing layer. Buccal devices were evaluated by different parameters such as weight uniformity, content uniformity, thickness, hardness, surface pH, swelling index, ex vivo mucoadhesive strength, ex vivo mucoadhesion time, in vitro drug release, and in vitro drug permeation. As compared with bilayered tablets, multilayered tablets showed slow release rate of drug with improved ex vivo bioadhesive strength and enhanced ex vivo mucoadhesion time. The mechanism of drug release was found to be non-Fickian diffusion (value of n between 0.5 and 1.0) for both the buccal devices. The stability of drug in both the optimized buccal devices was tested for 6 hours in natural human saliva; both the buccal devices were found to be stable in natural human saliva. The present study concludes that mucoadhesive buccal devices of PRH can be a good way to bypass the extensive hepatic first-pass metabolism and to improve the bioavailability of PRH. Published: March 16, 2007