Whole inactivated SIV vaccine grown on human cells fails to protect against homologous SIV grown on simian cells

Several groups have reported protection against experimental SIV infection in macaques immunized with a whole inactivated virus vaccine. The aim of the current study was to investigate whether five macaques vaccinated with whole inactivated SIV and previously shown to be protected against challenge with two divergent strains of SIV grown on human cells could resist challenge with a subsequent homologous SIV grown on macaque cells. We show here that this same vaccine did not protect when the challenge virus was grown on primary cells of monkey origin.     

Conjugative Plasmid Transfer between Bacteria under Simulated Marine Oligotrophic Conditions

Marine Vibrio S14 strains and an Escherichia coli strain were starved in artificial seawater (NSS) with no added carbon, nitrogen, or phosphorus. The broad-host-range plasmid RP1 was transferred between the starving S14 strains and also from the E. coli donor to the S14 recipient under oligotrophic conditions, in which mixtures of donor and recipient cells were held on Nuclepore filters either floated on NSS or held such that NSS flowed through the filter. Transconjugants were obtained from S14 donors and recipients starved for at least 15 days before being mixed together for conjugation, whereas transconjugants were recovered from the E. coli donor and S14 recipient for up to 3 days of prestarvation, but not after 5 days. Transconjugants were obtained when there were as few as about 10⁵ and 10⁴ cells of starving S14 donors and recipients, respectively, per ml held on the filters. Starved donor and recipient mixtures incubated at 4 or 26°C, as well as those allowed to mate for 2, 5, or 24 h, all yielded numbers of transconjugants which were not significantly (P > 0.05) different.     

Four centuries of cumulative impacts on a Finnish river and its estuary: an ecosystem health-approach

Changes in forest cover and draining of wetlands for agriculture appear to have caused changes in the aquatic ecosystem of the River Kyrönjoki by the 16th century. In the 19th century, a decline of salmonid fish populations was widely observed as a further sign of degradation. During the latter half of the present century intensified use of naturally acidic soils has resulted in increased acidic run-off. Deterioration of water quality has extended to the estuary, where it has caused large fish-kills and affected the reproductive success of coastal species. Degradation of the coastal ecosystem, first observed in the decline of salmonids and later as a more general decline of other coastal fish populations, can be linked to spatially restricted events. The loss of key river and estuarine habitats exerted an effect over the reproduction and abundance of species migrating throughout the system. This effect contributed to observed temporal and spatial discontinuities in the degradation history. Monitoring changes in critical habitats may prove to be an early indicator of changes in the health of estuarine and coastal aquatic ecosystems.     

Ependymal cells in tissue culture

Summary Ciliated ependymal cells from various locations of the ventricular system of mammals showed outgrowth either in the form of epithelial sheets or in the form of pools and elongated double rows, dependant on the site from which the ependyma was taken and on the original orientation of the cells in respect to the cover slip.The ependymal cilia in such cultures were shown to be moving at a rate of 51/2 to 6 times per second. The movement of the cilia of adjacent cells was apparently well coordinated causing the surrounding fluid to flow in a particular direction.The effect of physiological saline, morphine sulfate, ethyl and methyl alcohol on the ciliary motility has been studied in living cells with phase contrast cinematography.Grateful acknowledgement is made to Messers G. Lefeber, E. Pitsinger and J. Carnes for indispensible aid with photographic work. Mrs. J. Finerty and Mrs. W. Hild contributed generously in the management of the tissue cultures and in executing staining procedures.This investigation was supported by a research grant [PHS B-364 (C3)] from the National Institute of Neurological Diseases and Blindness, of the National Institutes of Health, Public Health Service, administered by C. M. Pomerat.     

The effect of ADF/cofilin and profilin on the dynamics of monomeric actin

The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

An amperometric cellobiose dehydrogenase-based biosensor can be used for measurement of cellulase activity

The hemoflavoenzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium has been used in an amperometric redox polymer-based biosensor. Used in conjugation with a FIA system this biosensor can replace colorimetric assays for measuring cellobiose liberated from cellulose in a series of cellulase-containing samples. The biosensor gave the same result as the Somogyi-Nelson method in a less time-consuming and laborious manner. The two methods showed about the same precision.     

The flexibility of actin filaments as revealed by fluorescence resonance energy transfer. The influence of divalent cations

The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f' is larger in calcium-F-actin than in magnesium-F-actin in the range of 5-40 degrees C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 degrees C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form.