Irreversible lysozyme inactivation and aggregation induced by stirring: kinetic study and aggregates characterisation

Stirring strongly enhanced irreversible inactivation and aggregation of lysozyme being studied as a model enzyme. From 0 to 740 rpm (equivalent to impeller tip speeds from 0 to 0.77 m s^–1), the inactivation kinetic constant was proportional to the power imparted by the impeller. Collisions between inactive and native molecules induced inactivation of the latter and led to lysozyme aggregation. These fractal aggregates of lysozyme were made of monomers, dimers and trimers.     

Diversity and evolution of coral fluorescent proteins

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.     

Characterization of the proteases involved in the N-terminal clipping of glucagon-like-peptide-1-antibody fusion proteins

In an attempt to develop high producing mammalian cell lines expressing glucagon-like-peptide-1-antibody fusion proteins (GLP-1), we have noted that the N-terminal GLP-1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N-terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine-threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products.     

Relationships Between Land Cover and Dissolved Organic Matter Change Along the River to Lake Transition

Dissolved organic matter (DOM) influences the physical, chemical, and biological properties of aquatic ecosystems. We hypothesized that controls over spatial variation in DOM quantity and composition (measured with DOM optical properties) differ based on the source of DOM to aquatic ecosystems. DOM quantity and composition should be better predicted by land cover in aquatic habitats with allochthonous DOM and related more strongly to nutrients in aquatic habitats with autochthonous DOM. Three habitat types [rivers (R), rivermouths (RM), and the nearshore zone (L)] associated with 23 tributaries of the Laurentian Great Lakes were sampled to test this prediction. Evidence from optical indices suggests that DOM in these habitats generally ranged from allochthonous (R sites) to a mix of allochthonous-like and autochthonous-like (L sites). Contrary to expectations, DOM properties such as the fluorescence index, humification index, and spectral slope ratio were only weakly related to land cover or nutrient data (Bayesian R ² values were indistinguishable from zero). Strongly supported models in all habitat types linked DOM quantity (that is, dissolved organic carbon concentration [DOC]) to both land cover and nutrients (Bayesian R ² values ranging from 0.55 to 0.72). Strongly supported models predicting DOC changed with habitat type: The most important predictor in R sites was wetlands whereas the most important predictor at L sites was croplands. These results suggest that as the DOM pool becomes more autochthonous-like, croplands become a more important driver of spatial variation in DOC and wetlands become less important.     

Epidemiology of the Hong Kong/68 Variant of Influenza A2 in Britain

Two influenza epidemics in Britain in 1968-9 and 1969-70, were due to the Hong Kong/68 variant of influenza A2 virus. The first epidemic was prolonged with low morbidity and mortality rates; the second was sharp with high rates. The difference between total morbidity and mortality in the two epidemics, however, was less than it appeared to be—the estimated excess morbidity and mortality due to all causes in 1969-70 was only about 50% greater than in 1968-9.Antibody studies showed that about one-quarter of two groups of adults investigated were infected in the first epidemic and about one-third in the second. After the two epidemics about one-third still had no antibody to the A2/Hong Kong/68 virus.     

Age-related changes in unscheduled DNA synthesis by rat hepatocytes

Ultraviolet-induced unscheduled DNA synthesis was studied as a function of age in hepatocytes isolated from 6- to 32-months-old rats. Unscheduled DNA synthesis was measured by both DNA specific activity and autoradiography. Using both procedures, a significant decline in unscheduled DNA synthesis was observed after 14 months of age.     

Induction of multiple forms of tyrosine aminotransferase by amino acid mixtures of different compositions

Hepatic tyrosine aminotransferase (EC 2.6.1.5) was induced in rats by intubation of amino acid mixtures (complete or tryptophan-free). Enzyme activity was increased 4-fold by the complete mixture and 8-fold by the tryptophan-free mixture. The enzyme was analyzed by chromatography on CM-Sephadex. Chromatographic patterns were characteristic of the type of inducer rather than of the chronology of the induction cycle: after induction by the complete amino acid mixture the three forms of the enzyme were equally increased whereas after induction by the tryptophan-free mixture Form I was preferentially increased.     

The fine structure of daphnid supraesophageal and optic ganglia,and its possible functional significance

Neurosecretory activity and fine structure of the supraesophageal and optic ganglia of Daphnia schødleri Sars were studied. The relative amount of paraldehyde fuchsin stainable material present was determined at “daylight” and at one and three hours following for animals maintained under photoperiods of 7.5, 10.5, 13.5, and 16.5 hours. More material was found after one hour in both ganglia and there was a tendency for more in the optic ganglion under 7.5- and 10.5-hour photoperiods. Sections made at two levels in the supraesophageal ganglion and one level in the optic ganglion were examined with an electron microscope. Posterior and anterior parts of the supraesophageal ganglion contain apparent nerve processes at the edge of the ganglion, parallel to the anterior-posterior axis; these have large granules. Neurons in both areas contain patches of presumed polysaccharide granules. In the posterior region are a dorsolateral and a ventrolateral glandular cell, presumably these occur on both sides of the brain. They have very well-developed endoplasmic reticulum and some large granules. The dorsal cell is usually at the tip of a glial attenuation. Concentric lamellar systems are located in dilated nerve processes of the first optic ganglion neuropile. Large whorls (about 3.5 μ) are composed of concentric lamellae. When lamellae do not form complete rings, they end in loops or in dilated tubules that are sometimes constricted as vesicles. Small whorls (1.5 μ) typically have lamellae joined into two or three thick layers. Mitochondria are frequently associated with the whorls. It is proposed that the whorls are active in synthesis, possibly neurohumor production.     

Alkylating ability of artemisinin after Cu(I)-induced activation

The reductive activation of artemisinin by copper(I)-dipyrrin or copper(I)-(2-Clip-Phen) complexes generates an artemisinin derived alkylating species leading to covalent artemisinin–copper complex adducts. The reactivity of the peroxide function of artemisinin toward Cu(I) complexes is similar to that of Fe(II) analogues, even though the reaction is more sluggish and product distribution slightly different.