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.     

Particle-Induced Pulmonary Acute Phase Response Correlates with Neutrophil Influx Linking Inhaled Particles and Cardiovascular Risk

Background

Particulate air pollution is associated with cardiovascular disease. Acute phase response is causally linked to cardiovascular disease. Here, we propose that particle-induced pulmonary acute phase response provides an underlying mechanism for particle-induced cardiovascular risk.

Methods

We analysed the mRNA expression of Serum Amyloid A (Saa3) in lung tissue from female C57BL/6J mice exposed to different particles including nanomaterials (carbon black and titanium dioxide nanoparticles, multi- and single walled carbon nanotubes), diesel exhaust particles and airborne dust collected at a biofuel plant. Mice were exposed to single or multiple doses of particles by inhalation or intratracheal instillation and pulmonary mRNA expression of Saa3 was determined at different time points of up to 4 weeks after exposure. Also hepatic mRNA expression of Saa3, SAA3 protein levels in broncheoalveolar lavage fluid and in plasma and high density lipoprotein levels in plasma were determined in mice exposed to multiwalled carbon nanotubes.

Results

Pulmonary exposure to particles strongly increased Saa3 mRNA levels in lung tissue and elevated SAA3 protein levels in broncheoalveolar lavage fluid and plasma, whereas hepatic Saa3 levels were much less affected. Pulmonary Saa3 expression correlated with the number of neutrophils in BAL across different dosing regimens, doses and time points.

Conclusions

Pulmonary acute phase response may constitute a direct link between particle inhalation and risk of cardiovascular disease. We propose that the particle-induced pulmonary acute phase response may predict risk for cardiovascular disease.     

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.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Quantification of indole-3-acetic acid from plant associated <Emphasis Type="Italic">Bacillus</Emphasis> spp. and their phytostimulatory effect on <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Sixteen Bacillus strains isolated from rhizosphere, histoplane and phyllosphere of different plant species were identified by 16S rDNA gene sequencing and evaluated for in vitro auxin production as well as growth stimulation of Vigna radiata (L.) Wilczek. Auxin production by Bacillus spp. in L-broth medium supplemented with 1,000 μg ml⁻¹ L-tryptophan ranges from 0.60 to 3.0 μg IAA ml⁻¹ as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. Rhizospheric isolates exhibit relatively more IAA synthesis than histoplane and phyllosphere isolates. Plant microbe interaction experiments conducted under gnotobiotic conditions recorded 55.55, 46.46 and 46.20% increase in shoot length with Bacillus megaterium MiR-4, B. pumilus NpR-1 and B. subtilis TpP-1, respectively, over control. Bacillus inoculations also increased shoot fresh weight with B. megaterium MiR-4 (60.94%) and B. pumilus NpR-1 (37.76%). Highly significant positive correlation between auxin production analyzed by GC–MS and shoot length (r = 0.687**, P = 0.01) and shoot fresh weight (r = 0.703**, P = 0.01) was noted under gnotobiotic conditions. Similarly, significant correlation was also found between auxin production by Bacillus spp. (GC–MS analysis) and different growth parameters such as shoot length (r = 0.495*, P = 0.05), number of pods (r = 0.498*, P = 0.05) and grain weight (r = 0.537*, P = 0.05) at full maturity under natural wire house conditions. Results showed that auxin production potential of plant associated Bacillus spp. can be effectively exploited to enhance the growth and yield of V. radiata.     

Structural dynamics of myoglobin: ligand migration and binding in valine 68 mutants

We have combined Fourier transform infrared/temperature derivative (FTIR-TDS) spectroscopy at cryogenic temperatures and flash photolysis at ambient temperature to examine the effects of polar and bulky amino acid replacements of the highly conserved distal valine 68 in sperm whale myoglobin. In FTIR-TDS experiments, the CO ligand can serve as an internal voltmeter that monitors the local electrostatic field not only at the active site but also at intermediate ligand docking sites. Mutations of residue 68 alter size, shape, and electric field of the distal pocket, especially in the vicinity of the primary docking site (state B). As a consequence, the infrared bands associated with the ligand at site B are shifted. The effect is most pronounced in mutants with large aromatic side chains. Polar side chains (threonine or serine) have only little effect on the peak frequencies. Ligands that migrate toward more remote sites C and D give rise to IR bands with altered frequencies. TDS experiments separate the photoproducts according to their recombination temperatures. The rates and extent of ligand migration among internal cavities at cryogenic temperatures can be used to interpret geminate and bimolecular O2 and CO recombination at room temperature. The kinetics of geminate recombination can be explained by steric arguments alone, whereas both the polarity and size of the position 68 side chain play major roles in regulating bimolecular ligand binding from the solvent.