Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest.

The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.     

Overexpression of Smad2 reveals its concerted action with Smad4 in regulating TGF-beta-mediated epidermal homeostasis.

Members of the transforming growth factor-beta (TGF-beta) superfamily are critical regulators for epithelial growth and can alter the differentiation of keratinocytes. Transduction of TGF-beta signaling depends on the phosphorylation and activation of Smad proteins by heteromeric complexes of ligand-specific type I and II receptors. To understand the function of TGF-beta and activin-specific Smad, we generated transgenic mice that overexpress Smad2 in epidermis under the control of keratin 14 promoter. Overexpression of Smad2 increases endogenous Smad4 and TGF-beta 1 expression while heterozygous loss of Smad2 reduces their expression levels, suggesting a concerted action of Smad2 and -4 in regulating TGF-beta signaling during skin development. These transgenic mice have delayed hair growth, underdeveloped ears, and shorter tails. In their skin, there is severe thickening of the epidermis with disorganized epidermal architecture, indistinguishable basement membrane, and dermal fibrosis. These abnormal phenotypes are due to increased proliferation of the basal epidermal cells and abnormalities in the program of keratinocyte differentiation. The ectodermally derived enamel structure is also abnormal. Collectively, our study presents the first in vivo evidence that, by providing an auto-feedback in TGF-beta signaling, Smad2 plays a pivotal role in regulating TGF-beta-mediated epidermal homeostasis.     

The 67-kDa laminin receptor originated from a ribosomal protein that acquired a dual function during evolution

The 67-kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that mediates high-affinity interactions between cells and laminin. Overexpression of this protein in tumor cells has been related to tumor invasion and metastasis. Thus far, only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated. The finding that the cDNA for the 37LRP is virtually identical to a cDNA encoding the ribosomal protein p40 has suggested that 37LRP is actually a component of the translational machinery, with no laminin-binding activity. On the other hand, a peptide of 20 amino acids deduced from the sequence of 37LR/p40 was shown to exhibit high laminin-binding activity. The evolutionary relationship between 23 sequences of 37LRP/p40 proteins was analyzed. This phylogenetic analysis indicated that all of the protein sequences derive from orthologous genes and that the 37LRP is indeed a ribosomal protein that acquired the novel function of laminin receptor during evolution. The evolutionary analysis of the sequence identified as the laminin-binding site in the human protein suggested that the acquisition of the laminin-binding capability is linked to the palindromic sequence LMWWML, which appeared during evolution concomitantly with laminin.      

Optimal range of prey size for antlions

1. Antlions are opportunistic trap building predators that cannot control prey encounter. Their trap should ideally retain a great diversity of prey. However, building a single trap that captures many prey with varying characteristics can be challenging. 2. A series of five different ant species ranging from thin to large, of sizes ranging from 2.75 to 6.5 mm, and a mean weight ranging from 0.54 to 6.00 mg were offered in a random succession to antlions. The state of satiation of the antlions was controlled, and their mass and the depth of their pit were recorded. The reaction of antlion to the prey, the probability of capture as well as the time to escape were recorded. 3. The probability of an antlion reaction is an increasing function of the pit depth and a decreasing function of antlion mass. The probability of capture is highest for intermediate prey mass and is an increasing function of pit depth. The time to escape is a declining function of prey mass and an increasing function of pit depth. 4. There is an upper limit to prey mass given that large prey escape out of the pit. There is a lower limit to prey mass given the difficulty to apprehend the smallest, thin species. Consequently, there is a range of prey mass, corresponding to a medium‐sized ant of 2 mg, for which the pit functions best. The physics of insect locomotion on sandy slopes was identified as the key to understanding the functioning of antlion pits.     

The calcium‐dependent protein kinase CPK7 acts on root hydraulic conductivity

The hydraulic conductivity of plant roots (Lp_r) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post‐translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp_r of knockout Arabidopsis plants for four Ca²⁺‐dependent protein kinases. cpk7 plants showed a 30% increase in Lp_r because of a higher aquaporin activity. A quantitative proteomic analysis of wild‐type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp_r of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7‐dependent stability of specific membrane proteins.     

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

Background

Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.

Results

The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.

Conclusions

A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.     

金属框架结构材料MOF-199对漆酶的固定化及其性质

以金属框架结构材料MOF-199为载体对漆酶进行固定化,并对固定化酶的性质进行初步研究。首先,以3-氨基丙基三乙氧基硅烷对载体MOF-199进行表面氨基化修饰,再用戊二醛对载体进行活化,最后对漆酶进行固定化。固定化条件优化结果表明:在漆酶质量浓度0.3 g/L,戊二醛用量1%(体积分数),pH 4.8下固定7 h,制得固定化酶活性最高。对固定化酶的研究发现:最适反应温度为40℃,最适pH为5.2,在连续操作7次后,固定化酶的活力仍能保持在51%。固定化漆酶热稳定性,pH耐受性,贮存稳定性均明显高于游离漆酶。