Systematic study of phospholipids linked to a steroid derivative, spread into a monolayer at the air/water interface.

Isothermal pressure-area curves of different phospholipids linked to a cortisol derivative, spread into monolayers at the air/water interface are studied. It is shown that derivatives containing saturated lipid chains and those with unsaturated chains present quite different behaviours. With saturated derivatives, the main phase transition plateau and the stability of the fluid phase are very sensitive to the length of the lipid chains, the presence of a spacer between the lipid and the steroid moieties, the temperature and the presence of di- and trivalent cations in the aqueous subphase; the calcium ion shows an especially high effect, compared to the other ions studied. The presence of the steroid on the lipid modifies the specific area of the molecules of unsaturated lipids, which is not the case with saturated lipids, probably due to differences in the lipophilic cohesion.

The interaction between lipid derivatives of colchicine and tubulin: consequences of the interaction of the alkaloid with lipid membranes.

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin-water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed.

Three-dimensional model of Escherichia coli gyrase B subunit crystallized in two-dimensions on novobiocin-linked phospholipid films.

Two-dimensional crystals of the Escherichia coli DNA gyrase B subunit were obtained upon specific interactions with novobiocin linked phospholipid films. A three-dimensional surface model of the protein was generated by analysing images of tilted negatively stained crystals. The structure showed, at 2.5 to 3.0 nm resolution, two elongated arms organised as a V-shaped protein: the bottom of the V contains the novobiocin binding site, and the extremities of the arms mediate protein-protein interactions between the two monomers in the unit cell. Image analysis of frozen hydrated two-dimensional crystals resulted in a 1.0 nm resolution projection map that shows structural elements not revealed with negative staining. Electron microscopic structural data were compared with the crystallographic structure of the 43 kDa N-terminal fragment of the B subunit complexed with a non hydrolysable ATP analogue.

Molecular organization of bovine rod cGMP-phosphodiesterase 6.

Phosphodiesterase 6 (PDE6), a multisubunit (alphabetagamma(2)delta) enzyme, plays a major role in visual function by hydrolysing cGMP in response to a light stimulus. Solubilized bovine rod PDE6 molecules depleted of their gamma subunits were purified to homogeneity from bovine retinal rods and their molecular organization was investigated by electron microscopy. Image analysis of single particles revealed the three-dimensional dimeric arrangement of the purified alphabetadelta complex, and the internal organization of each catalytic subunit into three distinct domains at a resolution of 2.8 nm. The relative volume of each domain is consistent with sequence analysis and functional data, which suggest that these domains correspond to the catalytic and two GAF domains. This hypothesis was confirmed by immunolabelling experiments, which located the N-terminal part of the catalytic subunit where the major interaction between the two alphabeta subunits was found to occur. The 3D molecular organization of human platelet PDE5 appears highly homologous to that of bovine rod PDE6, as predicted by similarities in their primary sequences. These observations describe the quaternary organization of the catalytic PDE6 alphabeta complex, and place the catalytic and regulatory domains on a structural model.

Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer.

Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.

Ligand- and DNA-induced dissociation of RXR tetramers.

Unliganded bacterially expressed RXR alpha lacking the N-terminal region AB (apo-RXR alpha delta AB) was found in solution as an apparent mixture of 165 kDa tetramers and 42 kDa monomers which could be quantitatively separated by gel filtration and non-denaturing gel electrophoresis. Under identical conditions both liganded (holo-) and apo-RAR alpha delta AB were present as single monomeric species. apo-RXR alpha delta AB tetramers, as well as dimers of the apo-RXR ligand binding domain (apo-LBD), dissociated readily into monomers when exposed to their cognate ligand 9-cis retinoic acid (9c-RA). The apo-RXR alpha delta AB tetramer bound only transiently to a cognate DR1 response element, and was converted into DR1-apo-RXR alpha delta AB homodimer complexes indistinguishable from those generated by cooperative DNA binding of apo-RXR alpha delta AB monomers. In the absence of DNA, the addition of 9c-RA greatly accelerated the formation of heterodimers with the apo-RAR alpha delta AB heterodimerization partner. No RXR alpha delta AB or RAR alpha delta AB homodimers could be observed in solution, but upon mixing of the two receptor monomers stable heterodimers could be isolated which bound to DR5 response elements in a highly cooperative manner. In these heterodimers, RXR alpha delta AB interacted with its cognate ligand as efficiently as in RXR alpha delta AB homodimers. The presence of ligand did not alter the stability of RXR alpha delta AB homodimer or RXR alpha delta AB-RAR alpha delta AB heterodimer complexes on DR1 and DR5 response elements, respectively. These in vitro data support a model in which RXR tetramers could serve as an inactive pool with the dual function of: (i) rapidly supplying large amounts of RXR heterodimerization partners upon 9c-RA generation; and (ii) allowing RXR homodimer formation on "accessible" cognate response elements in the absence of 9c-RA. These events may represent a ligand-dependent regulatory mechanism controlling the availability of the promiscuous RXR dimerization partner that is engaged in multiple nuclear receptor signalling pathways.

Two-dimensional crystallization of DNA gyrase B subunit on specifically designed lipid monolayers.

The B subunit of DNA gyrase formed two-dimensional crystals when bound to a specifically recognized phospholipid spread into a monolayer at the air/water interface. The especially designed lipids consisted of novobiocin coupled through the 3' or 2" hydroxyl group and a hydrophilous linker of a given length to dioleoylphosphatidic acid. Two-dimensional crystals of the gyrase B subunit are formed under physiological conditions of pH and ionic strength, with no precipitant added to the solution. Crystal diffraction extended to a 2.7 nm resolution in negative stain, with unit cell parameters a = 6.1 nm, b = 7.6 nm and gamma = 64 degrees.

Monitoring of intracellular levels of 5'-monophosphate-AZT using an enzyme immunoassay.

We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellular levels of 5'-monophosphate-AZT (AZT-MP). This assay is performed in 96-well microtiter plates coated with anti-rabbit immunoglobulin antibodies and is based on the use of rabbit polyclonal antibodies raised against an AZT-MP analog and of an AZT-MP/acetylcholinesterase conjugate as tracer. It is very sensitive, with a detection limit close to 0.1 ng/ml (0.2 pmol/ml), and precise (CV < 20% from 20 to 0.3 ng/ml). Very low cross-reactivities were observed with AZT and the corresponding di- and triphosphate derivatives as well as with other related nucleotides and nucleosides. The validity of the assay was demonstrated by measuring intracellular concentrations of AZT-MP in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived macrophages (MDMs) cultured in the presence of various concentrations of AZT (from 0.01 microM to 10 microM). We observed very high levels of AZT-MP in stimulated (PHA + IL2) PBMCs (> 100 pmol/10(6) cells) while, as expected, much lower concentrations were measured in resting PBMCs or MDMs (0.1 to 2 pmol/10(6) cells). The assay constitutes a very convenient tool permitting easy, precise studies of the first step of the intracellular metabolism of AZT leading to the formation of AZT-TP in cultured cells.

An unusual organism causing orbital cellulitis.

Bacterial orbital cellulitis is a feared complication of paranasal sinus infection. Staphylococcus and Streptococcus species are the commoner pathogens involved in these cases. However, anaerobic bacteria and unusual Gram-negative organisms should be suspected as well. We treated a case of bacterial orbital cellulitis due to foci of infected paranasal sinuses caused by Eikenella corrodens, a Gram-negative rod. The patient was managed with intensive antibiotic coverage and surgical intervention.

Synthesis of new fluidity-enhanced amphiphilic compounds for soluble protein two-dimensional crystallization purpose.

The synthesis of new amphiphilic compounds is described. The structures are rationally designed for soluble protein two-dimensional (2D) crystallization purpose. Special attention is devoted to fluidity properties expected of resulting monolayers. A series of 13 compounds was prepared containing unsaturated, branched or fluorinated alkyl chains. Structures are either symmetrical or dissymmetrical and present a hydroxyl group as polar head, eventually complemented with two other 'secondary' hydrophilic functions.

Rational design and synthesis of phospholipids for the two-dimensional crystallization of DNA gyrase, a key element in chromosome organization.

Properties required of lipids for two-dimensional crystallization of proteins on lipid layers at the air/water interface are discussed in terms of molecular structure. These properties are related to essential features of the overall system such as (i) the fluidity and stability of the lipid film, (ii) the affinity of the protein to be crystallized for the lipids and (iii) the accessibility of the protein to the ligand in the lipid layer as well as (iv) technical constraints of the crystallization technique. The resulting ideas were tested through the rational design and synthesis of original phospholipid structures linked to novobiocin subsequently used in the production of two-dimensional crystals of DNA gyrase (B subunit), a prokaryotic type II DNA topoisomerase.

Self-assembly of soluble proteins on functionalized lipid layers: a tentative correlation between the fluidity properties of the lipid film and protein ordering.

New series of amphiphilic structures are designed to exhibit various fluidity properties when spread at the air-water interface. The influence of the molecular structure of these lipids on the process of two-dimensional (2D) crystallization of the B subunit of DNA gyrase, a soluble protein, is investigated in terms of size of the crystals produced, protein ordering, and crystallization kinetics. Whereas no difference is observed concerning the mean size of the protein 2D crystals obtained on the different lipid supports, the ultimate protein ordering observable by electron microscopy using the negative-staining technique is more regularly attained with some of these new lipids. The most interesting point results from large discrepancies in crystallization kinetics as highly-ordered protein 2D crystals form within 6-24 h depending on the lipid layer structure. Thus, these new lipids reveal of special interest when studying proteins that suffer from extended incubation time at 4 degrees C or higher temperature and lose their functionality.

A selective and differential agar for anaerobic comma-shaped bacteria recovered from patients having motile rods and non-specific vaginosis.

A selective and differential agar for optimal growth of the large curved motile anaerobic rods isolated from patients having non-specific vaginosis (NSV) was developed. A basal medium of Columbia CNA agar was used with colony growth found to be optimal by the addition of 7% fetal calf serum and 5% rabbit blood (CNARS). Beta-Haemolysis was found to be demonstrated most readily on a bilayer plate with a basal layer consisting of CNA agar with fetal calf serum, overlaid with a layer of CNA agar containing fetal calf serum and rabbit blood. Optimal growth was found by anaerobic incubation at 37 degrees C, after 5 days.

[The computerized anesthesia record]. / La feuille d'anesthésie informatisée.

The first anaesthetic record was introduced into medical practice in 1940. Since then few changes have been made to it and it remains a rudimentary memorandum. However, since the beginning of the 1980s, interest in automatic recording of the anaesthetic file has been increasing and numerous arguments can be put forward in its favour. Apart from theoretical and experimental arguments, in practice one has to master the automatic collection of data, management of alarms and the technology of the networks involved in order to manage the flow of information by channelling it and organizing it into a hierarchy. Four other objectives can be added to the clinical recording and its medico-legal applications: anaesthetic cost evaluation, quality of care, research and clinical teaching which will provide the basis of anaesthetic epidemiological research.

Complications of methylene blue dye in breast surgery: case reports and review of the literature.

INTRODUCTION: Methylene blue dye has been used worldwide successfully with few complications in breast surgery. We present two different complications involving methylene blue: 1) skin and parenchymal necrosis when dye was injected in a subdermal fashion and 2) Mycoplasma infection caused by contaminated methylene blue in breast reduction surgery. METHODS: We present two cases seen at the University of Arizona during 2008 and referred to a breast surgeon for management. We evaluated and managed complications of methylene blue dye injected by 2 referring surgeons for different indications. A review of the literature was performed. RESULTS: The first case is a 67 year old female diagnosed with infiltrating ductal carcinoma of the left breast for which she was treated by her initial surgeon with left segmental mastectomy and sentinel node biopsy. The operating surgeon injected methylene blue in a subareolar subdermal fashion (distant from the primary tumor); unfortunately the patient suffered skin and breast necrosis requiring multiple surgical debridements and finally achieving delayed primary closure. The second case is a 45 year old female with infiltrating lobular carcinoma with a history of Mycoplasma infection secondary to methylene blue injected for breast reduction surgery. She required multiple debridements and had granulomas masquerading as cancer on MRI that confounded her extent of disease. CONCLUSIONS: The use of methylene blue dye in breast surgery is not without risk. In both cases methylene blue was responsible for complications requiring surgical debridement for local wound problems. In each case severe necrosis and infection were present. Methylene blue may cause not only significant morbidity, but may also produce cosmetically unsatisfactory results.

Roots of automation in microbiology: an introduction.

It seems like a long way from the early work of Pasteur and Koch to the automated microbiology laboratory of today. But every advance rests on a knowledge base gleaned from past work; and to understand where we are today, it is necessary to know how we got there. This paper explores the historical roots of current microbiology technology.

Short-range specific forces are able to induce hemifusion.

Working with pure lipidic systems (giant unilamellar vesicles, 10-150 microm in diameter) as models for biological membranes, we have considered possible structures of the contact area of two adherent membranes by investigating the diffusion of fluorescent lipid analogues from one vesicle to another. Two bilayers in close contact can almost be seen as a lamellar structure in equilibrium. This is the usual configuration of two adherent vesicles, in which the interbilayer distance is estimated to be 3 nm. We have increased the attraction between the membranes by either adding depletion forces or by using a trick, inspired from the interaction between nucleic bases in nucleosides (herein adenosine and thymidine). The nucleosides were attached to the polar head of amphiphilic molecules that behave like phospholipids and were incorporated in the model membrane. The extra attraction between two membranes, resulting from base pairing, strongly decreased the interbilayer distance down to about 1 nm. This change of the water content induced lipid rearrangements, which could also be viewed in terms of a phase transition at low water content. These rearrangements were not observed in the case of depletion forces. We conclude that the introduction of an additional attractive force in the system modifies the equilibrium state, leading to a drastic change in the membrane behavior, which will tentatively be related to hemifusion.