Analysis of excited-state processes by phase-modulation fluorescence spectroscopy

Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-stale protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (I) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (φ) larger than 90° or by measurement of ratios of m/cos φ > 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overtap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene(PRODAN) and N-acetyl-l-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos φ measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.     

Electron microscopic and cytochemical studies of rat aorta. Intracellular vesicles containing elastin- and collagen-like material

Synopsis Small, rounded vesicles with a dense core of amorphous material were observed in all cell types in the young rat aorta, that is, endothelial cells, smooth muscle cells and fibroblasts. They were particularly numerous in the Golgi complex but were also found in the cell periphery. The content of the vesicles had staining characteristics identical to those of elastin. Material of the same type was also found in cisternae on the maturing side of the dictyosomes and in vesicles budding from them. Reaction product for thiamine pyrophosphatase was present in both these structures, indicating that the Golgi complex is responsible for the formation of the dense-cored vesicles. This was further supported by the absence of reaction product for acid phosphatase in the cisternae and in the vesicles. Moreover, no uptake of exogenous markers was noted in the latter. On the basis of these findings it is suggested that the dense-cored vesicles have a secretory function and contain precursors of elastin.Elongated vesicles or profiles containing collagen fibrils were observed in smooth muscle cells and fibroblasts. In the cell periphery, these vesicles were often found to communicate with the extracellular space. Further inside the cells, they showed a close spatial relationship to the Golgi complex. Neither thiamine pyrophosphatase nor acid phosphatase activity was demonstrated in the elongated vesicles. Like the plasma membrane, their limiting membrane was positively stained for alkaline phosphatase. On the basis of these findings and the absence of uptake of exogenous markers in them, it is suggested that the elongated vesicles represent a means for collagen secretion in the growing aortic wall. The Golgi complex is believed to be involved in the transfer of collagen to these vesicles.     

Meiofaunal (re)colonization of the Arctic intertidal (Hornsund,Spitsbergen) after ice melting: role of wrack deposition

Intertidal meiobenthos of Hornsund—the southernmost fjord of Spitsbergen—was investigated between July and September 2005. Mean total meiofaunal densities ranged between 4.3 and 328 ind. 10 cm⁻². Nematode assemblages were impoverished in terms of the number of genera when compared with those from the western Spitsbergen coast (11 vs. 25–28 genera in total, respectively). It is suggested that severe environmental conditions in the southern part of Spitsbergen overcome the adaptation skills of many nematode species and hamper the establishment of a diverse community. Comparatively high nematode numbers on a beach subject to heavy macroalgal wrack input contrast sharply with numerically poor communities in sparse-wrack beaches (up to 315 vs. 31 ind. 10 cm⁻², respectively). It is suggested that the wrack input to the Arctic beach may substantially influence the richness and composition of the intertidal meiobenthic community. Nematode assemblages were dominated by extreme colonizers: Geomonhystera disjuncta and rhabditids. Their relatively high densities in beach sediments recorded at the beginning of July indicate their ability to recover rapidly after the winter period and to effective (re)colonization of the intertidal habitat just after the ice melt. Average concentration of rhabditids and monhysterids associated with macroalgal wrack deposited on the upper shore was as high as 52 × 10³ individuals per gram of the substrate. It is hypothesized that nematodes can play a substantial role in the wrack decomposition in the Arctic intertidal.     

Specificity of Endo-β-1,3-glucanase G A from Cellulomonas cellulans towards Structurally Diversified Acceptor Molecules in Transglycosylation Rreaction

The synthetic properties of homogeneous endo- &#103 -1,3-glucanase G A from Cellulomonas cellulans were studied. Thirty-one synthetic and natural &#102 - and &#103 -glycosides were examined as acceptor molecules in transglycosylation reactions conducted in an aqueous-organic solvent environment with &#103 -1,3-glucan as a polymeric donor. Seventeen acceptors underwent significant glycosylation. The best acceptors for glucanase G A were &#102 - or &#103 -glycosides that, in the favoured chair conformation of the pyranose ring, had all the equatorial substituents. Glycosides having an axial 4-hydroxyl or 2-hydroxyl group in the pyranose ring represent a poor class of acceptors.     

Molecular aspects of bacterial nanocellulose biosynthesis

Bacterial nanocellulose (BNC) produced by aerobic bacteria is a biopolymer with sophisticated technical properties. Although the potential for economically relevant applications is huge, the cost of BNC still limits its application to a few biomedical devices and the edible product Nata de Coco, made available by traditional fermentation methods in Asian countries. Thus, a wider economic relevance of BNC is still dependent on breakthrough developments on the production technology. On the other hand, the development of modified strains able to overproduce BNC with new properties – e.g. porosity, density of fibres crosslinking, mechanical properties, etc. – will certainly allow to overcome investment and cost production issues and enlarge the scope of BNC applications. This review discusses current knowledge about the molecular basis of BNC biosynthesis, its regulations and, finally, presents a perspective on the genetic modification of BNC producers made possible by the new tools available for genetic engineering.     

Nature and the Multiple Functions of the 67-kD Elastin-/Laminin Binding Protein

Numerous cell types, including fibroblasts, vascular smooth muscle cells, chondroblasts, monocytes, neutrophils, and several tumor cells express the 67-kD galactolectin, homologous to the alternatively spliced variant of β-galactosidase. The 67-kD protein resides on the cell surfaces and is capable of interacting with elastin, laminin and collagen type IV. This peripheral membrane protein binds its matrix ligands but only in the absence of galactosugars, whereas binding of galactosugar-containing moieties to its lectin site changes its molecular folding which causes discharge of the ligand and release of the receptor from the cell surface. This review will address the functional significance of the single receptor that interacts with multiple matrix proteins and can be shed from cell surfaces by galactosugars. I will emphasize the role of the 67-kD protein in divergent cellular processes, such as cell-matrix attachment, matrix assembly, cellular chemotaxis, and active migration through the vascular walls.     

Cloning and expression of Thiobacillus versutus cryptic plasmid pTAV-1 DNA in Escherichia coli

pTAV-1 is an approximately 100 kb Thiobacillus versutus cryptic plasmid. pTAV-1 DNA was cloned in Escherichia coli. Nine recombinant plasmids containing pTAV-1 DNA inserted into the EcoRI restriction site of pACYC184 were constructed. The origin of DNA inserts was confirmed by Southern blot hybridization. The expression of mixotrophic T. versutus plasmid genes was demonstrated in E. coli.