Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.     

Gibbon ape leukemia virus receptor functions of type III phosphate transporters from CHOK1 cells are disrupted by two distinct mechanisms

The Chinese hamster cell lines E36 and CHOK1 dramatically differ in susceptibility to amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GALV); E36 cells are highly susceptible to both viruses, CHOK1 cells are not. We have previously shown that GALV can infect E36 cells by using both its own receptor, HaPit1, and the A-MuLV receptor, HaPit2. Given that the two cell lines are from the same species, the loss of function of both of these receptors in CHOK1 cells is surprising. Other studies have shown that CHOK1 cells secrete proteins that block A-MuLV entry into CHOK1 as well as E36, suggesting the two A-MuLV receptors are functionally identical. However, CHOK1 conditioned medium does not block GALV entry into E36, indicating the secreted inhibitors do not block HaPit1. HaPit1 and ChoPit1 therefore differ as receptors for GALV; ChoPit1 is either inactivated by secreted factors or intrinsically nonfunctional. To determine why GALV cannot infect CHOK1, we cloned and sequenced ChoPit1 and ChoPit2. ChoPit2 is almost identical to HaPit2, which explains why CHOK1 conditioned medium blocks A-MuLV entry via both receptors. Although ChoPit1 and HaPit1 are 91% identical, a notable difference is at position 550 in the fourth extracellular region, shown by several studies to be crucial for GALV infection. Pit1 and HaPit1 have aspartate at 550, whereas ChoPit1 has threonine at this position. We assessed the significance of this difference for GALV infection by replacing the aspartate 550 in Pit1 with threonine. This single substitution rendered Pit1 nonfunctional for GALV and suggests that threonine at 550 inactivates ChoPit1 as a GALV receptor. Whether native ChoPit1 functions for GALV was determined by interference assays using Lec8, a glycosylation-deficient derivative of CHOK1 that is susceptible to both viruses and that has the same receptors as CHOK1. Unlike with E36, GALV and A-MuLV exhibited reciprocal interference when infecting Lec8, suggesting that they use the same receptor. We conclude both viruses can use ChoPit2 in the absence of the inhibitors secreted by CHOK1 and ChoPit1 is nonfunctional.     

Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.     

Novel halo-oxo-allenic fatty ester derivatives from epoxidized methyl santalbate (methyl trans-11-octadecen-9-ynoate)

Methyl santalbate (methyl trans-11-octadecen-9-ynoate) from Sandal wood seed oil, Santalbum alum) was epoxidized to methyl trans-11,12-epoxy-octadec-9-ynoate (1). Treatment of compound 1 with tetrabutylammonium dihydrogentrifluoride, and boron trifluoride etherate gave the corresponding anti- (2a) (57%) and syn- (2b) (35%) fluorohydrin derivatives, respectively. These reactions were regio- and stereoselective in nature. The structures of the anti- and syn- isomers were confirmed by NMR spectroscopy. Ring opening of the epoxy system of compound 1 with lithium chloride gave the anti-chlorohydrin derivative (3) (89%). Oxidation of either compound 2a or 2b gave the same fluoro-keto acetylenic fatty ester (4) (75%), and compound 3 on chromic acid oxidation yielded the corresponding chloro-keto acetylene (5) (73%). Isomerization of compounds 4 and 5 with potassium carbonate in dichloromethane furnished the requisite fluoro-allenic (6) (63%, methyl 11-fluoro-12-oxo-9,10-octadecadienoate) and chloro-allenic (7) (80%, methyl 11-chloro-12-oxo-9,10-octadecadienoate) C(18) fatty esters. All products were confirmed by a combination of spectrometric and spectroscopic techniques.     

Cardiac sympathetic afferent stimulation impairs baroreflex control of renal sympathetic nerve activity in rats

It is well known that cardiac sympathetic afferent reflexes contribute to increases in sympathetic outflow and that sympathetic activity can antagonize arterial baroreflex function. In this study, we tested the hypothesis that in normal rats, chemical and electrical stimulation of cardiac sympathetic afferents results in a decrease in the arterial baroreflex function by increasing sympathetic nerve activity. Under alpha-chloralose (40 mg/kg) and urethane (800 mg/kg i.p.) anesthesia, renal sympathetic nerve activity, mean arterial pressure, and heart rate were recorded. The arterial baroreceptor reflex was evaluated by infusion of nitroglycerin (25 microg i.v.) and phenylephrine (10 microg i.v.). Left ventricular epicardial application of capsaicin (0.4 microg in 2 microl) blunted arterial baroreflex function by 46% (maximum slope 3.5 +/- 0.3 to 1.9 +/- 0.2%/mmHg, P < 0.01). When the central end of the left cardiac sympathetic nerve was electrically stimulated (7 V, 1 ms, 20 Hz), the sensitivity of the arterial baroreflex was similarly decreased by 42% (maximum slope 3.2 +/- 0.3 to 1.9 +/- 0.4%/mmHg; P < 0.05). Pretreatment with intracerebroventricular injection of losartan (500 nmol in 1 microl of artificial cerebrospinal fluid) completely prevented the impairment of arterial baroreflex function induced by electrical stimulation of the central end of the left cardiac sympathetic nerve (maximum slope 3.6 +/- 0.4 to 3.1 +/- 0.5%/mmHg). These results suggest that the both chemical and electrical stimulation of the cardiac sympathetic afferents reduces arterial baroreflex sensitivity and the impairment of arterial baroreflex function induced by cardiac sympathetic afferent stimulation is mediated by central angiotensin type 1 receptors.     

Elevation of plasma phospholipid transfer protein in transgenic mice increases VLDL secretion

Two lipid transfer proteins are active in human plasma, cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP). Mice by nature do not express CETP. Additional inactivation of the PLTP gene resulted in reduced secretion of VLDL and subsequently in decreased susceptibility to diet-induced atherosclerosis. The aim of this study is to assess possible effects of differences in PLTP expression on VLDL secretion in mice that are proficient in CETP and PLTP. We compared human CETP transgenic (huCETPtg) mice with mice expressing both human lipid transfer proteins (huCETPtg/huPLTPtg). Plasma cholesterol in huCETPtg mice was 1.5-fold higher compared with huCETPtg/huPLTPtg mice (P < 0.001). This difference was mostly due to a lower HDL level in the huCETPtg/huPLTPtg mice, which subsequently could lead to the somewhat decreased CETP activity and concentration that was found in huCETPtg/huPLTPtg mice (P < 0.05). PLTP activity was 2.8-fold increased in these animals (P < 0.001). The human PLTP concentration was 5 microg/ml. Moderate overexpression of PLTP resulted in a 1.5-fold higher VLDL secretion compared with huCETPtg mice (P < 0.05). The composition of nascent VLDL was similar in both strains. These results indicate that elevated PLTP activity in huCETPtg mice results in an increase in VLDL secretion. In addition, PLTP overexpression decreases plasma HDL cholesterol as well as CETP.     

β-Oxidation capacity in liver increases during parr-smolt transformation of Atlantic salmon fed vegetable oil and fish oil

Atlantic salmon Salmo salar were fed diets containing 100% fish oil (FO; capelin oil) or 100% vegetable oil (VO) from start of feeding until the fish reached the size of 2·5 kg. Samples were taken during the period of the parr-smolt transformation (October 2002 to February 2003). The VO diet consisted of a blend of 55% rapeseed oil, 30% palm oil and 15% linseed oil to maintain the sum of saturated, monounsaturated and polyunsaturated fatty acids between the two diets, although with differences in the individual chain length of fatty acids. Na⁺/K⁺-ATPase activity in the gills, total β-oxidation capacity in muscles and liver and total lipid, glycogen and dry matter content in the muscles were measured during the parr-smolt transformation and after seawater transfer. Na⁺/K⁺-ATPase activity in gills increased prior to seawater transfer, showing an adaptation for seawater survival. Major changes in the lipid and glycogen content in the fillet and in β-oxidation capacity were found in the tissues measured. β-oxidation capacity increased significantly in liver and decreased in red muscle, prior to seawater transfer, giving liver an important role in energy production during this period. Results also indicated that feeding Atlantic salmon a diet where 100% of FO was replaced with VO did not have any negative effects on lipid metabolism during parr-smolt transformation.     

Characterization and implications of host-cell protein aggregates in biopharmaceutical processing

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.     

棉蚜体色变化的生态遗传学研究

调查了不同寄主上棉蚜刀Aphis gossypll自受精卵孵化出的自然种群、室内混合饲养以及单个饲养蚜虫的体色变化。结果表明：不论是自然还是实验种群,是群体还是个体饲养,不论寄主、栽培条件、生育期营养相同与否,棉蚜体色在世代内稳定不变,即出生时是什么颜色保持终生不变;在世代间则随温度升高体色渐变为黄色,温度降低体色逐渐转绿。伏蚜由苗蚜而来。X²检验证实：棉蚜体色变化与营养、寄主种类、光照、光质、栽培条件等无关,仅与温度密切相关,属于同一基因型在不同环境条件下的反应规范。但在太槿上还发现有个别深黄色棉蚜,从卵孵化到迁飞体色不随温度变化,表明棉蚜体色变化中还存在遗传多态现象。胚胎学观察与染色体校型分析结果证实了上述结论与观点。     

农药醚菊酯类似物构效关系的人工神经网络方法研究

本文以醚菊酯类似物作为研究对象,尝试使用神经网络方法进行构效关系分析,并对该种农药活性进行了预测。在所研究的样本集中,由结构预测活性的成功率可达100％,本文的研究表明：神经网络方法以其极强的非线性能力,可望成为农药构效关系研究的一种有效的工具．