Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z_eff) and nonelectrostatic (i.e., specific) component of binding, K_d(1M). Gag binds to Psi RNA with a dramatically reduced K_d(1M) and lower Z_eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z_eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z_eff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.     

Cobblestone HUVECs: a human model system for studying primary ciliogenesis

Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium.In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear.Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body.We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.     

Alterations in the muscle-to-capillary interface in patients with different degrees of chronic obstructive pulmonary disease

Background

It is hypothesized that decreased capillarization of limb skeletal muscle is implicated in the decreased exercise tolerance in COPD patients. We have recently demonstrated decreased number of capillaries per muscle fibre (CAF) but no changes in CAF in relation to fibre area (CAFA), which is based on the diffusion distance between the capillary and muscle fibre. The aim of the current study is to investigate the muscle-to-capillary interface which is an important factor involved in oxygen supply to the muscle that has previously been suggested to be a more sensitive marker for changes in the capillary bed compared to CAF and CAFA.

Methods

23 COPD patients and 12 age-matched healthy subjects participated in the study. Muscle-to-capillary interface was assessed in muscle biopsies from the tibialis anterior muscle using the following parameters:1) The capillary-to-fibre ratio (C:F_i) which is defined as the sum of the fractional contributions of all capillary contacts around the fibre2) The ratio between C:F_i and the fibre perimeter (CFPE-index)3) The ratio between length of capillary and fibre perimeter (LC/PF) which is also referred to as the index of tortuosity.Exercise capacity was determined using the 6-min walking test.

Results

A positive correlation was found between CFPE-index and ascending disease severity with CFPE-index for type I fibres being significantly lower in patients with moderate and severe COPD. Furthermore, a positive correlation was observed between exercise capacity and CFPE-index for both type I and type IIa fibres.

Conclusion

It can be concluded that the muscle-to-capillary interface is disturbed in the tibialis anterior muscle in patients with COPD and that interface is strongly correlated to increased disease severity and to decreased exercise capacity in this patient group.     

Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

O-GalNAc-glycosylation is one of the main types of glycosylation in mammalian cells. No consensus recognition sequence for the O-glycosyltransferases is known, making prediction methods necessary to bridge the gap between the large number of known protein sequences and the small number of proteins experimentally investigated with regard to glycosylation status. From O-GLYCBASE a total of 86 mammalian proteins experimentally investigated for in vivo O-GalNAc sites were extracted. Mammalian protein homolog comparisons showed that a glycosylated serine or threonine is less likely to be precisely conserved than a nonglycosylated one. The Protein Data Bank was analyzed for structural information, and 12 glycosylated structures were obtained. All positive sites were found in coil or turn regions. A method for predicting the location for mucin-type glycosylation sites was trained using a neural network approach. The best overall network used as input amino acid composition, averaged surface accessibility predictions together with substitution matrix profile encoding of the sequence. To improve prediction on isolated (single) sites, networks were trained on isolated sites only. The final method combines predictions from the best overall network and the best isolated site network; this prediction method correctly predicted 76% of the glycosylated residues and 93% of the nonglycosylated residues. NetOGlyc 3.1 can predict sites for completely new proteins without losing its performance. The fact that the sites could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc.     

Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles

PDC-109 is the major protein of bovine seminal plasma. It binds to the bovine sperm surface at ejaculation and modulates sperm capacitation. PDC-109 displays phosphorylcholine- and heparin-binding activities which are thought to account for its sperm surface coating and glycosaminoglycan-induced sperm capacitating activities, respectively. We have characterized the interaction of isolated PDC-109 with membranes of phospholipid vesicles using a biophysical approach. Our results show that PDC-109 interacts not only with the solvent-exposed phosphorylcholine head group but also with the hydrophobic core of liposomes. Binding of PDC-109 to membranes is a very rapid, biphasic process with half times of less than one second. Maximal binding of PDC-109 to small unilamellar vesicles was achieved with a stoichiometric ratio of 10–11 phosphatidylcholine molecules/PDC-109 molecule. Incorporation of phosphatidylethanolamine or phosphatidylserine into phosphatidylcholine vesicles reduced the binding of PDC-109, suggesting that both the density of phosphorylcholine groups and the surface charge determine the interaction of the seminal plasma protein with the surface of the membrane. Electron spin resonance measurements showed that binding of PDC-109 to phosphatidylcholine vesicles caused a rigidification of the membrane. The relevance of the data for describing the role of PDC-109 in the modulation of sperm capacitation is discussed. Received: 16 June 1997 / Accepted: 10 September 1997     

Growth and cellular organization of Arabidopsis pollen tubes in vitro

Tricellular pollen tubes of Arabidopsis thaliana were cultured in vitro on solid media and studied with respect to growth, cellular organization and ultrastructure, cytoskeletal organization, organelle movement, deposition and structure of the wall and the occurrence of coated pits, all elements assumed to be relevant for tip growth. For our ultrastructural studies we used freeze fixation and freeze substitution. Although Arabidopsis pollen tubes are broadly similar to those of bicellular species such as Nicotiana tabacum and Lilium spec. and in vivo grown pollen tubes of Arabidopsis, some differences occurred. The density of the equally distributed, relatively small (85 nm) secretory vesicles (SV) in the tip is low (five/µm ²). In between the SV of the tip, membranous material, possibly smooth endoplasmic reticulum, fragments of rough endoplasmic reticulum and loose ribosomes are present. The wall in the tip is not amorphous but layered and a secondary wall is formed already in the flanks of the tip. The general pattern of organelle motion is reverse fountain-like, but individual organelles move in distinct lanes at speeds of up to 2 µm/s, and about half of the organelle population shows a moderate velocity or Brownian movement. These properties are discussed in relation to the low growth rate (10 µm/h) of Arabidopsis pollen grown in vitro. The two similar sperm cells are closely attached and are always found near the vegetative nucleus. No surrounding wall and no cytoskeletal elements were obvious in the sperm cells. The preferential location of the mitochondria at the wall and the large (up to 400 nm) coated pits are unique for angiosperm pollen tubes. The size of the coated pits may allow not only membrane retrieval but also pinocytosis.