The "beta-clasp" model of apolipoprotein A-I -- a lipid-free solution structure determined by electron paramagnetic resonance spectroscopy

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Two different schemes of twice-weekly ovum pick-up in dairy heifers: effect on oocyte recovery and ovarian function

The aim of the present study was to compare two different schemes of twice-weekly ovum pick-up (OPU), continuous (C) and discontinuous (DC), with special emphasis on differences in oocyte yield and quality, estrous cyclicity, ovarian dynamics, and progesterone production. Subsequent to characterization of their normal estrous cycles (pre-OPU period), eight dairy heifers were subjected to 4 months of twice-weekly OPU under two different schemes: the DC (OPU restricted to Days 0-12 of the cycle) and the C schemes. Effects of the two different schemes on oocyte yield, quality, and in vitro competence, together with effects on ovarian dynamics and progesterone production, were monitored. The mean numbers of punctured follicles and recovered oocytes per session were slightly higher (not significant (n.s.)) using the DC scheme, but in total, similar numbers of oocytes were obtained. The quality of the oocytes as well as cleavage rate after in vitro fertilization of the oocytes did not differ between the two OPU schemes. There was no influence of a corpus luteum (CL) producing progesterone on the oocyte yield and quality, whereas the presence of dominant follicles appeared to decrease the number of recovered ooctyes. During the pre-OPU period, all heifers showed normal cyclicity. In the DC scheme, the heifers showed regular and normal cyclic activity throughout the puncture period, with one to two complete follicular waves during the interval from the last OPU to the next estrus. In the C scheme, the heifers occasionally revealed cyclicities with irregular interestrous intervals and weaker signs of estrus. No complete follicular waves were seen during the OPU period in this scheme. The CL developed from the ovulation of the preovulatory follicles in the DC scheme showed similar characteristics to the CLs of the pre-OPU period; however, the CL-like structures from the puncture of follicles, in both the DC and the C schemes, revealed a shorter life span and inferior competence in producing progesterone (P<0.05). The present results indicate that the DC OPU scheme, which allows animals to go into natural ovulation prior to the first OPU, does not affect their ovarian function, whereas the C OPU scheme does. Our study further demonstrates that an equal number of oocytes can be obtained with both schemes, but that fewer OPUs are needed when the DC scheme is applied.     

Some characteristic reactions catalysed by the ribonuclease activity in sections from fixed tissues

Summary 1. The ribonuclease activity in sections from freeze-dried Carnoy-fixed rat pancreas has been characterized through studies on the products of interaction with synthetic nucleoside derivatives, nucleotide esters, and of the polynucleotide synthetic properties.2. Homogenates of the sections in McIlvaine's buffer atph 7.0 degrade pyrimidine 2:3-phosphates to the corresponding 3-dihydrogen phosphate, and the purine derivatives at a much slower rate.3. Synthetic cytidylyl-cytidine, and benzyl- and ethyl-3-esters of pyrimidine nucleotides are degraded to nucleoside 3-phosphates. All the purine, and the 2-esters of pyrimidine nucleotides are not affected during 24 hours of incubation.4. Under influence of the homogenates, ribopolynucleotides will readily be synthesized from cytidine-2:3-phosphate and cytidine, the 3-P-5dinucleotide links being identified in cytidylyl-cytidine and cyclic dicytidylic acid.5. The conclusion is reached that the activity of the sections coincides with the DEase and CPase activity of crystalline pancreatic RNase. Some aspects of the possibilities for the histochemical localization of the activity are briefly discussed.With 6 Figures in the TextAided by grants from the Royal Physiographic Society of Lund, the Nora Sörensson Foundation, and the J. V. and Charlotta Lundgren Foundation.     

Structure and function of the GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae

The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Deltagtr1 similar to the Deltapho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84-green fluorescent protein or Pho84-myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1-185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186-310) displayed no evidence of conformational changes upon GTP addition.     

Structural modeling of dual-affinity purified Pho84 phosphate transporter

The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.     

Association in multifactorial traits: how to deal with rare observations?

To detect the role of a candidate gene for a trait in a sample of individuals, we may test SNP haplotype or diplotype effects. For a limited sample size, many haplotype or diplotype categories may contain few individuals. This involves a power decrease when testing the association between the trait and the haplotypes or diplotypes as these categories provide little additional information while increasing the degrees of freedom. The present paper proposes a new strategy to group rare categories based on a measure of similarity between haplotypes or diplotypes and compares it to two other possible strategies to deal with rare categories: a SNP selection strategy based on haplotype diversity, and a grouping strategy that pools all rare categories into a single baseline group. This comparison is performed by means of simulation under four scenarios. We show that this new strategy shows the largest increase in power irrespective of the model underlying the candidate gene in the studied trait. This strategy therefore provides a powerful alternative to currently used methods to reduce the number of rare categories.     

The effect of formaldehyde containing fixatives on ribonuclease activity

Summary 1. The inactivation of crystalline ribonuclease by formaldehyde and formaldehyde containing fixatives (Serra's solution) is demonstrated.2. The rate of inactivation is shown to be dependent uponph, formaldehyde concentration, and time of action of the fixative.3. The effect of formaldehyde containing fixatives on the RNase activity in sections from fixed tissues is discussed, and the inactivation of that enzyme system in rat pancreas is demonstrated.With 2 Figures in the Text     

Translocation of radioactive kinetin

Kinetin has generally been thought to be immobile in plants. This was confirmed in the case of laminar applications in this study, but not in regard to petiole, vein, or root applications. Radioactivity from kinetin-8-¹⁴C (Kn^*) moved freely in the vascular system of several types of leaves. This movement was usually distal to the point of application and seemed to occur with the transpiration stream. Basipetal as well as acropetal translocation of radioactive kinetin was achieved in tobacco leaves. The translocated material was extracted from veinal tissue, shown to be radioactive, and to be able to retard senescence. Similar but less decisive results were obtained from agar blocks inserted into the vascular system of leaves receiving Kn^* by petiole uptake.

A bioassay employing disks from primary bean leaves was developed for the qualitative determination of substances like kinetin which possess the ability to retard chlorophyll breakdown and plant senescence. The use of radioactive kinetin provided a refinement in this bioassay because treated non-senescent areas could be correlated with exposed areas on radioautographs made from dried leaf disks.

Root treatments showed that cotton seedlings did not take up Kn^* but that similarly treated tobacco seedlings both absorbed and translocated the isotope readily.