Isolation of a novel receptor tyrosine kinase cDNA expressed by developing erythroid progenitors

Activation of distinct receptor tyrosine kinases (RTK), such as the products of the c-fms and c-kit proto-oncogenes, profoundly affects hematopoietic development. We have isolated a novel RTK cDNA, called met-related kinase (MRK), which is expressed on early erythroid progenitors. MRK is also expressed in many hematopoietic cell lines, and is not lineage restricted. Several regions within the catalytic domain of MRK have amino acid similarity to the c-met proto-oncogene and v-sea oncogene. Specific polyclonal anti-MRK antisera detects an 84- Kd polypeptide in COS cells transfected with an expression vector containing the MRK cDNA. Further studies of this novel RTK will provide potential insight into its role during hematopoiesis.     

Skeletally anchored forsus fatigue resistant device for correction of Class II malocclusions—A systematic review and meta-analysis

The use of skeletal anchorage with fixed functional appliances (FFA) has been proposed by various authors to produce skeletal changes and reduce lower incisor proclination. To compare the skeletal and dentoalveolar effects of Forsus Fatigue Resistant Device (FFRD) with or without skeletal anchorage (miniplates and mini-implants). The electronic database PubMed, Cochrane Library, Medline, Embase and Google Scholar along with a manual search of orthodontic journals till the year 2019. Only randomized control trials (RCTs) were included in the systematic review. One controlled clinical trial (CCT) which involved FFRD was included in the review since it was a continuation of an RCT which was expanded to a CCT. Skeletal and dentoalveolar outcome data were extracted to collect study characteristics. After evaluating risk of bias, the standardized mean differences (SMDs) and 95% confidence intervals (CIs) were calculated. Three RCTs and one prospective CCT were evaluated. The analysis included data from 116 Class II subjects [(58) treated with FFA along with skeletal anchorage and (58) treated with FFA]. There were no significant difference between the two groups with respect to mandibular length changes (P value = .10) and SNB angle changes (P value = .22). With respect to lower incisor inclination however, there was a significant difference between the two groups (P value = .005) signifying better results with respect to skeletal anchorage. The studies reviewed provide insufficient evidence to form a conclusion regarding the effects of the use of skeletal anchorage with FFRD. The available weak evidence suggests that the use of skeletal anchorage with FFRD has no superior skeletal effects but is able to reduce proclination of the lower incisors. Control of lower incisor proclination remains the most significant advantage of skeletal reinforcement, and miniplate-anchored FFRD showed more promising results in preventing lower incisor proclination than miniscrew-anchored FFRD.     

Detergent-free isolation,characterization, and functional reconstitution of a tetrameric K+ channel: The power of native nanodiscs

A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or “native nanodiscs.” Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid–protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general.Integral membrane proteins (MPs) are an abundant class of proteins that play key roles in a wide range of essential cellular processes (1). To facilitate their study in vitro, detergent molecules are commonly used to extract MPs out of their native lipid–bilayer environment (2). However, the use of detergents has some inherent disadvantages. Most importantly, even though there are promising developments to improve their properties (3, 4), the insufficient mimicking of a lipid bilayer by detergent micelles often leads to destabilization and rapid loss of function of the incorporated protein (5). For many functional and structural studies, it is thus necessary to reconstitute the MP into a more stabilizing environment; for example, by replacing the detergent with amphipathic polymers (amphipols) (6) or incorporating the MP into lipid nanodiscs with a surrounding protein scaffold (7). Both approaches have proven to be valuable tools for the study of structural and functional properties of MPs (8, 9); however, a limitation remains, as transfer of MPs into any of these systems requires initial solubilization by detergent.Recently, a detergent-free approach has been described using amphipathic styrene-maleic acid copolymers (SMAs) as an alternative to solubilize MPs directly from biological membranes in the form of nanodiscs, referred to as “Lipodisq” or SMA lipid particles (10–13) (Fig. 1). The mechanism of action of SMA differs fundamentally from that of detergents: instead of disrupting the lipid bilayer completely, SMA spontaneously self-inserts and extracts intact membrane patches in the form of discoidal particles that are stabilized by a SMA annulus (14, 15). Because these nanodiscs conserve a spatially delimited native biomembrane including MPs, we term them “native nanodiscs.” One of the main advantages of this system is the straightforward extraction protocol without the need for detergent. It has been shown that the SMA polymer is capable of directly extracting native nanodiscs containing large functional protein complexes from yeast (12), bacterial proteins involved in cell division (16) and photosynthesis (17), and several members of the ABC transporter family (13). The isolation of these proteins from a variety of different organisms suggests a general applicability of SMA solubilization for all MPs, irrespective of their expression host or native organism.Open in a separate windowFig. 1.(A) Chemical structure of SMA polymers at neutral pH. For this study, a polymer with an average SMA ratio of n:m = 2:1 was used. (B) Schematic representation of a native nanodisc containing a KcsA tetramer (blue) and native lipids (green). The outer hydrophobic surface of the lipids is shielded by SMA (orange).To further explore the potential of native nanodiscs, we used the SMA polymer to isolate an oligomeric bacterial membrane protein: the tetrameric potassium channel from Streptomyces lividans (KcsA) (18), expressed in Escherichia coli. KcsA is an ideal model protein for such studies because it is well-characterized and because reconstitution studies have shown that both its function and stability are strongly affected by lipid composition (19–21). In this work, we apply SMA to prepare and purify native nanodiscs with KcsA to compare the conformational properties and stability of the protein with those in detergent micelles. In addition, we use native nanodiscs to investigate preferential lipid–protein interactions by analyzing the composition of small patches of native membrane that are copurified with the protein. Finally, we study the functional properties of KcsA on reconstitution from native nanodiscs into a planar lipid bilayer system. Our results underscore the huge potential of SMA as a membrane-solubilizing agent, as well as the use of native nanodiscs as a membrane-mimetic system for biophysical studies on ion channels, and MPs in general.     

A non-flow cytometric system for detecting antibodies and cellular antigens in blood

The system described here can distinguish between single and agglutinated erythrocytes by use of a non-flow fiber optic fluorometer. The method is capable of detecting cell-surface antigens and antibodies to cell-surface antigens present in blood. Significant features include high-efficiency fluorescent dyes that intercalate into cell membranes, a stretching membrane for transport and mixing of samples, charged colloidal magnetite for magnetic separation of erythrocytes, and an immersible fiber optic probe for measuring fluorescence associated with cells in a 1-nL volume of a bulk solution. We describe the application of the system to automation of ABO/Rh grouping and antibody screening.     

茜草中萘酸酯类成分的研究

从茜草根中分离出四个萘酸甲酯化合物。经光谱分析及化学方法鉴定,化合物Ⅰ为一新成分,命名为茜草内酯(rubilactone,Ⅰ),化合物Ⅱ～Ⅳ分别为已知物3’-甲氧羰基-4’-羟基-萘骈[1’,2’-2,3]呋喃(furomollugin,Ⅱ),二氢大叶茜草素(dihydromollugin,Ⅲ),2-(3’-羟基)异戊基-3-甲氧羰基-1,4-萘氢醌-1-O-β-D-吡喃葡萄糖甙(Ⅳ)。其中Ⅱ系自茜草属中首次分得。     

Spermatic vein embolization with hot contrast material: fertility results

Spermatic venography with hot contrast material embolization was undertaken in 81 patients with varicoceles and infertility. Long-term follow-up information was available in 91% of the patients, and there was an overall conception rate of 40.5%. Embolization with hot contrast material was easily performed without special embolization devices and proved to be a safe and effective technique.     

禁食和再喂养期间大鼠小肠胰岛素和胰岛素样生长因子-1受体的比较

胰岛素样生长因子-1（IGF-1）和胰岛素可能是肠道生长的重要调节因子。为了研究肠细胞萎缩和再生期间小肠IGF－1受体（IGF-IR）和胰岛素受体（IR），我们比较了禁食72小时和肠内再喂养24～72小时大鼠空肠IGF-IR和IR表达的指标。禁食引起肠萎缩，血浆胰岛素和IGF－1浓度降低以及空肠IGF-1信使RNA（mRNA）水平的明显降低，再喂养可逆转这些改变。禁食明显增加胰岛素与空肠特异性地结合，IR含量（达对照组的230％）和9．6kb和7．4kbIRmRNA转录本水平（分别达对照组的202％和218％）。再喂养时，这些IR指标迅速降到对照组水平。禁食时IGP-IR（用Scatchard分析）和IGF－1－RmRNA无明显的改变。再喂养后的前24小时间11-kbIGF－IRmRNA转录本明显增加（达对照组水平166％），IGF-IR数量增加3倍。我们的结论是：大鼠空肠的IR和IGF－IR受到不同营养物利用状态的调节。再喂养时空肠IGF－1和IGF－IR表达的向上调节表明，IGF作用途径在对肠内营养物产生肠道营养反应的过程中起作用。