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PubMed2006

Self-assembly of fatty acid-alkylboladiamine salts.

Long-chain fatty acids are insoluble in aqueous solution and form crystal precipitates. It is then of particular importance to determine the physicochemical parameters allowing their dispersion in water to improve their bioavailability and their utilization as surfactants. Herein, we report a study on salt-free catanionic systems in aqueous solution made of mixtures between palmitic or stearic fatty acids and alkylboladiamines (Abd's) differing by their alkyl chain length. Phase contrast microscopy, solid-state NMR, Fourier transform infrared spectroscopy, and small-angle neutron scattering were used to characterize the phase behavior of these systems at molar ratio of fatty acid to Abd of 1 and 2. Whatever the Abd and the molar ratio, fatty acids were embedded at low temperature in a bilayer gel phase which crystallizes after a period of rest. At an equimolar ratio, the gel phases transited upon raising the temperature to an isotropic phase made of worm-like micelles except in the case of the ethylenediamine chain for which a lamellar fluid phase was observed. At a molar ratio of 2 and high temperature, fatty acids were embedded in a lamellar fluid phase which self-orients with its stacking axis perpendicular to the magnetic field. However, for a long alkylboladiamine such as spermine, worm-like micelles formed. The phase behavior at high temperature is discussed in terms of molecular volume.

Alkanes↗
PubMed2003

Diacylglycerol-rich domain formation in giant stearoyl-oleoyl phosphatidylcholine vesicles driven by phospholipase C activity.

We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (alpha-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles. Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy. After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering. These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries. Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs.

Diglycerides↗
PubMed2005

Composition dependence of vesicle morphology and mixing properties in a bacterial model membrane system.

We have determined the mixing properties and lamellar organization of bacterial membrane mimetics composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol (POPG) at various molar ratios applying differential scanning calorimetry, small and wide-angle X-ray scattering, as well as optical phase contrast microscopy. Combining the experimental thermodynamic data with a simulation of the liquidus and solidus lines, we were able to construct a phase diagram. Using this approach, we find that the lipids mix in all phases non-ideally in the thermodynamic sense. As expected, pure POPE assembles into multilamellar and pure POPG into unilamellar vesicles, respectively, which are stable within the studied temperature range. In contrast, mixtures of the two components form oligolamellar vesicles consisting of about three to five bilayers. The layers within these oligolamellar liposomes are positionally correlated within the gel phase, but become uncorrelated within the fluid phase exhibiting freely fluctuating bilayers, while the vesicles as a whole remain intact and do not break up into unilamellar forms. X-ray, as well as DSC data, respectively, reveal a miscibility gap due to a lateral phase segregation at POPG concentrations above about 70 mol%, similar to previously reported data on mixtures composed of disaturated PEs and PGs. Hence, the existence of a region of immiscibility is a general feature of PE/PG mixtures and the mixing properties are dominated by PE/PG headgroup interactions, but are largely independent of the composition of the hydrocarbon chains. This is in accordance with a recent theoretical prediction.

Bacteria↗
PubMed1966

Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope.

Razin, Shmuel (University of Connecticut, Storrs), and Benjamin J. Cosenza. Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope. J. Bacteriol. 91:858-869. 1966-Growth of 11 Mycoplasma strains in liquid media was followed by phase-contrast microscopy. A similar pattern of development was common to all strains. Branching filaments, 0.3 to 0.4 mu thick, characterized the early logarithmic phase of growth. The length of the filaments varied according to the strain tested and the growth medium. Addition of oleic acid to the medium induced the formation of very long filaments by M. laidlawii strain B. Upon aging, the filaments were found to break up into chains of coccoid elements. These chains further fragmented to yield shorter chains and single coccoid elements, which characterized the stationary and decline phases of growth. The size of the coccoid elements increased from 0.3 to 0.4 mu, when formed in the filaments, to 0.6 to 0.8 mu after being released from the chains. Further increase in the size of the cells took place at the decline phase of growth, leading to the formation of very large cells reaching a diameter of 10 to 20 mu. However, these large cells had the appearance of empty vesicles and were apparently nonviable as indicated by viable-count experiments.

Culture Media↗
PubMed1991

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: light and electron microscopic study.

The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.

Adult↗
PubMed1980

Changes in cell diameter during the division cycle of Escherichia coli.

Extensive measurements of steady-state populations of several Escherichia coli strains have consistently indicated that cell diameter decreases with increasing cell length. This was observed both after electron microscopy of air-dried cells and after phase-contrast microscopy of living cells. The analysis was made by considering separately the unconstricted cells and three classes (slight, medium, and deep) of constricted cells in the population. During slow growth, cells with the average newborn length were up to 8% thicker than unconstricted cells twice as long. This decrease in diameter is less at higher growth rates. Despite the small changes and the large variation of the diameter in any particular length class, significant negative correlations between diameter and length were obtained. Cell diameter increases again at the end of the cell cycle as indicated by an increase of average diameter in the three consecutive classes of constriction.

Bacillus subtilis↗
PubMed2004

Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations.

BACKGROUND: We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. METHODS: We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. RESULTS: There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, approximately 68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n = 367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. CONCLUSIONS: Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI.

Cell Shape↗
PubMed1987

Comparison of a DNA-DNA dot-blot hybridisation assay with light microscopy and radioimmunoassay for the detection of a nuclear polyhedrosis virus.

A dot-blot hybridisation assay was developed for the detection of a nuclear polyhedrosis virus (NPV) and was compared to light microscopy and radioimmunoassay (RIA). Using cloned NPV DNA labelled with 32P as a probe, a number of hybridisation assay procedures was examined. The assay was found to be more sensitive than differential staining, phase-contrast microscopy, or indirect solid-phase RIA with as few as 20 occlusion bodies (150 pg DNA) being detected. Samples do not require prior purification or DNA extraction. The assay was shown to be specific for NPV and has the potential to detect and discriminate between strains of the virus. With little modification the assay may be used to detect other insect viruses.

Animals↗
PubMed1989

Dysmorphism of urinary red blood cells--value in diagnosis.

To aid investigation into the clinical problem of hematuria, assessment of abnormalities in the shape of red cells in the urine (dysmorphism) is gaining popularity in nephrology. However, there is uncertainty in the literature regarding both the number of red blood cells (RBC) in normal urine, as well as the quantification of dysmorphism. We have shown that in normal urine (N = 27) the number of RBC is less than 2,000/ml as assessed by scanning electron microscopy of filtered urine specimens from normal volunteers without known renal disease, which compared to less than 1,000/ml by centrifugation and phase contrast microscopy of the same specimen. To determine whether dysmorphism of urinary red blood cells was a significant predictor of glomerular disease we compared the number of dysmorphic cells in the urine of patients with biopsy proven glomerulonephritis (GN), before and immediately after renal biopsy. We also compared the number of dysmorphic cells in patients with glomerulonephritis to those with lower urinary tract bleeding. Renal biopsy caused significant dysmorphic hematuria, indicating that dysmorphism suggests renal rather than glomerular bleeding. Although patients with GN had significantly more dysmorphic urinary RBC when compared to those with lower tract urinary bleeding, the overlap was such that one could only be confident of renal hematuria if they accounted for greater than 75% of the total number of RBC. Non renal hematuria is present if number of dysmorphic cells is less than 17% of total RBC. Thus dysmorphism of urinary RBC is a useful diagnostic tool, but only if strict criteria established for each laboratory are adhered to.

Erythrocyte Count↗
PubMed1988

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy.

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.

Bacteroides↗
PubMed2003

Assembly of collagen matrices as a phase transition revealed by structural and rheologic studies.

We have studied the structural and viscoelastic properties of assembling networks of the extracellular matrix protein type-I collagen by means of phase contrast microscopy and rotating disk rheometry. The initial stage of the assembly is a nucleation process of collagen monomers associating to randomly distributed branched clusters with extensions of several microns. Eventually a sol-gel transition takes place, which is due to the interconnection of these clusters. We analyzed this transition in terms of percolation theory. The viscoelastic parameters (storage modulus G' and loss modulus G") were measured as a function of time for five different frequencies ranging from omega = 0.2 rad/s to 6.9 rad/s. We found that at the gel point both G' and G" obey a scaling law, with the critical exponent Delta = 0.7 and a critical loss angle being independent of frequency as predicted by percolation theory. Gelation of collagen thus represents a second order phase transition.

Animals↗
PubMed2000

Dynein and dynactin deficiencies affect the formation and function of the Spitzenkörper and distort hyphal morphogenesis of Neurospora crassa.

The impact of mutations affecting microtubule-associated motor proteins on the morphology and cytology of hyphae of Neurospora crassa was studied. Two ropy mutants, ro-1 and ro-3, deficient in dynein and dynactin, respectively, were examined by video-enhanced phase-contrast microscopy and image analysis. In contrast to the regular, hyphoid morphology of wild-type hyphae, the hyphae of the ropy mutants exhibited a great variety of distorted, non-hyphoid morphologies. The ropy hyphae were slow-growing and manifested frequent loss of growth directionality. Cytoplasmic appearance, including organelle distribution and movement, were ostensibly different in the ropy hyphae. The Spitzenkörper (Spk) of wild-type hyphae was readily seen by phase-contrast optics; the Spk of both ro-1 and ro-3 was less prominent and sometimes undetectable. Only the fast-growing ropy hyphae displayed a Spk, and it was smaller and less phase-dark than the wild-type Spk. Growth rate in both wild-type and ropy mutants was directly correlated with the size of the Spk. Spk efficiency, measured in terms of cell area generated per Spk travelled distance, was lower in ropy mutants. Another salient difference between ropy mutants and wild-type hyphae was in Spk trajectory. Whereas the Spk of wild-type hyphae maintained a trajectory close to the cell growth axis, the Spk of ropy hyphae moved much more erratically. Sustained departures in the trajectory of the ropy Spk produced corresponding distortions in hyphal morphology. A causal correlation between Spk trajectory and cell shape was tested with the Fungus Simulator program. The characteristic morphologies of wild-type or ropy hyphae were reproduced by the Fungus Simulator, whose vesicle supply centre (VSC) was programmed to follow the corresponding Spk trajectories. This is evidence that the Spk controls hyphal morphology by operating as a VSC. These findings on dynein or dynactin deficiency support the notion that the microtubular cytoskeleton plays a major role in the formation and positioning of the Spk, with dramatic consequences on hyphal growth and morphogenesis.

Computer Simulation↗
PubMed1972

Physical studies of isolated eucaryotic nuclei.

The degree of chromatin condensation in isolated rat liver nuclei and chicken erythrocyte nuclei was studied by phase-contrast microscopy as a function of solvent pH, K(+) and Mg(++) concentrations Data were represented as "phase" maps, and standard solvent conditions selected that reproducibly yield granular, slightly granular, and homogeneous nuclei Nuclei in these various states were examined by ultraviolet absorption and circular dichroism (CD) spectroscopy, low-angle X-ray diffraction, electron microscopy, and binding capacity for ethidium bromide Homogeneous nuclei exhibited absorption and CD spectra resembling those of isolated nucleohistone. Suspensions of granular nuclei showed marked turbidity and absorption flattening, and a characteristic blue-shift of a crossover wavelength in the CD spectra. In all solvent conditions studied, except pH < 2 3, low-angle X-ray reflections characteristic of the native, presumably superhelical, nucleohistone were observed from pellets of intact nuclei. Threads (100-200 A diameter) were present in the condensed and dispersed phases of nuclei fixed under the standard solvent conditions, and examined in the electron microscope after thin sectioning and staining Nuclei at neutral pH, with different degrees of chromatin condensation, exhibited similar binding capacities for ethidium bromide. These data suggest a model that views chromatin condensation as a close packing of superhelical nucleohistone threads but still permits condensed chromatin to respond rapidly to alterations in solvent environment.

Animals↗