Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release

Evanescent-wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass-water interface on top of which the cells are grown. Whereas epi-illumination images appeared blurred and structureless, evanescent-wave excitation highlighted acridine orange-labelled vesicles as individual pinpoints. Three-dimensional (3D) trajectories of individual vesicles were obtained from time-resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D(3). Based on the single-vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [K+]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking.     

PEG as a tool to gain insight into membrane fusion

Thirty years ago, Klaus Arnold and others showed that the action of PEG in promoting cell–cell fusion was not due to such effects as surface absorption, cross-linking, solubilization, etc. Instead PEG acted simply by volume exclusion, resulting in an osmotic force driving membranes into close contact in a dehydrated region. This simple observation, based on a number of physical measurements and the use of PEG-based detergents that insert into membranes, spawned several important areas of research. One such area is the use of PEG to bring membranes into contact so that the role of different lipids and fusion proteins in membrane fusion can be examined in detail. We have summarized here insights into the fusion mechanism that have been obtained by this approach. This evidence indicates that fusion of model membranes (and probably cell membranes) occurs via severely bent lipidic structures formed at the point of sufficiently close contact between membranes of appropriate lipid composition. This line of research has also suggested that fusion proteins seem to catalyze fusion in part by reducing the free energy of hydrophobic interstices inherent to the lipidic fusion intermediate structures. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

A new insight into the pathogenesis of filarial disease

Filariasis is a major public health problem throughout many regions of the tropics. The disease is caused by several species of filarial nematode including Wuchereria bancrofti and Brugia malayi, the agents of lymphatic filariasis, and Onchocerca volvulus, the cause of 'riverblindness'. Disease caused by these worms varies depending on the tissue location of the parasite, and is associated with episodes of acute and chronic inflammation. These pathologies, including elephantiasis and blindness, rank among the most disabling in the world. Studies aimed at characterizing the molecular nature of the inflammatory stimuli derived from filarial nematodes uncovered a long forgotten secret, their symbiont Wolbachia. LPS-like molecules from these intracellular bacteria are responsible for potent inflammatory responses from macrophages and in animal models of filarial disease. Wolbachia has also been associated with severe inflammatory reactions to filarial chemotherapy, being released into the blood following the death of the parasite. Recent studies in animal models even implicate Wolbachia in the onset of lymphodema and blindness. Taken together these studies suggest a major role for Wolbachia in the pathogenesis of filarial disease. It may be possible, through the use of antibiotic therapy, to clear worms of their bacteria, in the hope that this will prevent the onset and development of filarial pathology.     

Tackling the human adipose tissue proteome to gain insight into obesity and related pathologies

Obesity is becoming an important public health problem given its strong association with insulin resistance and Type 2 diabetes. Previously considered an inert depot, fat is now regarded as a highly metabolically active tissue in many pathophysiological processes. In humans, the accumulation of omental rather than subcutaneous adipose tissue appears to be tightly linked to cardiovascular disease and other important comorbidities. Proteomics has emerged as a method for the large-scale study of proteins in biological samples, for instance, fluids, cells or tissues, which encompasses not only the identities of the proteins present, but also quantification and post-translational modification events. Human adipose tissue proteome analysis, still in its early stages, may help understand the molecular mechanisms of obesity and the role of omental fat in the pathogenesis of obesity-associated diseases. This review covers recent advances in human adipose tissue proteomics, focusing on the analysis of the omental and the subcutaneous fat.     

A new insight into mercurized hemoglobin aggregation mechanism

Coagulation of bovine oxyhemoglobin in the presence of mercuric acetate in concentrations within a range including concentrations exceeding those required to block the single pair of thiol groups of the protein has been investigated in Tris-acetate buffer. The values of initial coagulation rate plotted against mercury-to-hemoglobin molar ratio give curves exhibiting a clear break points at ratios corresponding to full blocking of the mentioned thiol groups. Larger amounts of mercury reagents producing enhanced protein coagulation effect depend approximately quadratically on the mercury concentration. Interaction of the excess mercuric ions with some mercury-binding sites located on or near the dimer-dimer contact surfaces of the protein producing stronger coagulation effect is suggested.     

A new insight into the gelatinization process of native starches

The gelatinization characteristics of seven different food starches (regular corn, high-amylose corn, waxy corn, wheat, rice, potato, and tapioca) were investigated. Each starch sample type was heated to 35, 40, 45, etc. up to 85 °C at 5 °C intervals, and freeze-dried. The treated samples were analyzed using light microscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and high-performance size exclusion chromatography (HPSEC). When heated, granules underwent structural changes prior to the visible morphological changes that took place during gelatinization. The nature of these structural changes depended on starch type. These results indicate that the starch gelatinization process is more complex than a simple granular order-to-disorder transition.     

Proteomic analysis provides new insight into the chicken eggshell cuticle

The cuticle is the outermost layer of the avian eggshell, whose protein constituents remain virtually unknown. We hypothesize that cuticle components play a major role in microbial resistance, since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination. In this study we extracted proteins from the outermost non-calcified layer of the cuticle of chicken eggs and subjected them to LC/MS/MS proteomic analysis. We identified 47 cuticle proteins with high confidence and reproducibility. Two proteins, similar to Kunitz-like protease inhibitor and ovocalyxin-32 (a carboxypeptidase A inhibitor), were the most abundant of the cuticle proteins. A number of proteins known to have antimicrobial activity in the egg were detected (lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor) as well as possible new candidates (myeloperoxidase, ovocalyxin-36 and members of the SERPIN family). This is the first comprehensive report of cuticle proteome, a starting point to determine cuticle function and the molecular basis of its antimicrobial properties.     

A new insight into solid-state conformation of macrolide antibiotics

Quantitative analysis of the molecular conformations of the 14-membered macrolide antibiotics erythromycin A and B, clarithromycin, and roxithromycin in the solid state was performed. While the erythronolide macrocycle adopts a very similar folded-out conformation in all the macrolides studied, the proximity of the monosaccharide moieties, L-cladinose and D-desosamine, to each other is demonstrated to be the distinctive feature of their molecular conformations, based on atom-atom interaction energy analysis. More surprisingly, the common features in the relative orientation of the monosaccharide moieties (in terms of non-bonded atom-atom interactions) were revealed between the 14- and 15-membered (azithromycin) macrolide antibiotics. Herein we report on the details of the spatial arrangement of the monosaccharide moieties in these structurally related drug molecules and their influence on the biopharmaceutical properties of erythromycin derivatives.     

Genosystematics and new insight into the phylogeny and taxonomy of liverworts

The current state of molecular studies in liverworts, including original data, was considered. The traditional concepts of the liverwort phylogeny and systematics have greatly changed as a result of recent molecular researches. The phylogenetic inferences from studies of different DNA loci of different species sampling are mainly congruent. The phylogeny and systematics of the suborder Jungermaniineae, one of the largest and taxonomically difficult groups, is discussed on the basis of nucleotide sequence analyses of internal transcribed spacers 1 and 2 (ITS1-2) of nuclear rDNA and chloroplast trnL-F in a representative species sampling.     

Compressibility gives new insight into protein dynamics and enzyme function

The adiabatic compressibility of enzyme is largely influenced by binding of coenzyme and substrate, due to the changes in atomic packing. Amino acid substitution also induces large changes in compressibility parallel to enzyme activity. These results demonstrate that a small alteration of local structure by ligand binding and mutation is dramatically magnified in the flexibility of protein molecule to affect the function. Compressibility gives new insight into protein dynamics and enzyme function from the aspect of atomic packing or cavity which cannot be obtained by other techniques.     

Sponge genes provide new insight into the evolutionary origin of the neurogenic circuit

The nerve cell is a eumetazoan (cnidarians and bilaterians) synapomorphy [1]; this cell type is absent in sponges, a more ancient phyletic lineage. Here, we demonstrate that despite lacking neurons, the sponge Amphimedon queenslandica expresses the Notch-Delta signaling system and a proneural basic helix loop helix (bHLH) gene in a manner that resembles the conserved molecular mechanisms of primary neurogenesis in bilaterians. During Amphimedon development, a field of subepithelial cells expresses the Notch receptor, its ligand Delta, and a sponge bHLH gene, AmqbHLH1. Cells that migrate out of this field express AmqDelta1 and give rise to putative sensory cells that populate the larval epithelium. Phylogenetic analysis suggests that AmqbHLH1 is descendent from a single ancestral bHLH gene that later duplicated to produce the atonal/neurogenin-related bHLH gene families, which include most bilaterian proneural genes [2]. By way of functional studies in Xenopus and Drosophila, we demonstrate that AmqbHLH1 has a strong proneural activity in both species with properties displayed by both neurogenin and atonal genes. From these results, we infer that the bilaterian neurogenic circuit, comprising proneural atonal-related bHLH genes coupled with Notch-Delta signaling, was functional in the very first metazoans and was used to generate an ancient sensory cell type.     

A new insight into cattle's maternal origin in six Asian countries

The domestication of cattle fuelled the development of agricultural society in the history of human being.The evolution and genetic relationship of cattle can be elucidated by investigating the variation of mitochondrial DNA(mtDNA) D-loop sequence.In this study,we built a cattle phylogeny with a pool of 856 individual D-loop sequences,of which 264 Chinese cattle D-loop sequences were obtained in this study(141 ones were first analyzed,and 123 were first submitted) and the rest sequences of cattle from six A...     

A new insight into cattle's maternal origin in six Asian countries

The domestication of cattle fuelled the development of agricultural society in the history of human being.The evolution and genetic relationship of cattle can be elucidated by investigating the variation of mitochondrial DNA (mtDNA) D-loop sequence.In this study,we built a cattle phylogeny with a pool of 856 individual D-loop sequences,of which 264 Chinese cattle D-loop sequences were obtained in this study (141 ones were first analyzed,and 123 were first submitted) and the rest sequences of cattle from six Asian countries (Japan,Korea,Mongolia,Nepal,India and China) were retrieved from GenBank.Our results indicated that cattle from six Asian countries fell into three clades,Bos taurus (taurine),Bos indicus (zebu) and yak.Four main haplogroups T1A,T2,T3 (including T3A and T3B) and T5 were found in taurine,and two haplogroups I1 and I2 in zebu.Furthermore,we found that I1 and I2 haplogroups were separated by four variable sites rather than five ones and four haplogroups or sub-haplogroups of T1A,T3A,T3B and T5 were found for the first time in these Asian cattle.These data brought us a new insight into cattle's genetic structure in these six Asian countries.The geographical distribution of haplogroups was also outlined to provide systematic information on cattle genetic resources.     

Solid state NMR: new tools for insight into enzyme function

NMR has had considerable impact in enzymology, probing evidence for ionization states, conformational 'strain', compressed interactions, electronically unusual species, and conformational dynamics of enzymes. Solid-state NMR is becoming increasingly important in studying enzymes because of a number of recent tools for analysis of proteins by SSNMR, and because of the growing ability to isolate the species of interest for analysis. Here, we review recent studies of a Michaelis complex, of the dynamic functioning of membrane-associated enzymes, and initial studies of several enzymes with redox-active and paramagnetic centers.     

Exploring residues crucial for nitrilase function by site directed mutagenesis to gain better insight into sequence-function relationships

Nitrilases represent a very important class of enzymes having an array of applications. In the present scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A, Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and pattern of nitrile hydrolysis.