Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine FcγR Types

We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.     

Growth and phylogenetic properties of novel bacteria belonging to the epsilon subdivision of the Proteobacteria enriched from Alvinella pompejana and deep-sea hydrothermal vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45 degrees C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Colour discrimination in dim light by the larvae of the African catfish <Emphasis Type="Italic">Clarias gariepinus</Emphasis>

Many demersal fish species undergo vertical shifts in habitats during ontogeny especially after larval metamorphosis. The visual spectral sensitivity shifts with the habitat, indicating a change in colour vision. Colour vision depends on sufficient ambient light and becomes ineffective at a particular low light intensity. It is not known how fishes see colour in dim light. By means of a behavioural experiment on larval African catfish Clarias gariepinus in the laboratory, we determined colour vision and colour discrimination in dim light. Light-adapted larvae were subjected to classical conditioning to associate a reward feed with a green or a red stimulus placed among 7 shades of grey. The larvae learned this visual task after 70 and 90 trials. A different batch of larvae were trained to discriminate between green and red and then tested for the ability to discriminate between these colours, as the light intensity was reduced. The larvae learned this visual task after 110 trials in bright light and were able to discriminate colours, as light was dimmed until 0.01 lx, the minimal illuminance measurable in this study, and similar to starlight. The retinae of the larvae were found to be light adapted at 0.01 lx; thus indicating cone-based colour vision at this illuminance. For comparison, three human subjects were tested under similar conditions and showed a colour vision threshold at between 1.5 and 0.1 lx. For the larvae of C. gariepinus, the ability of colour discrimination in dim light is probably due to its retinal tapetum, which could increase the sensitivity of cones.     

The effects of CDP-choline on newborn rat pups with experimental alcohol fetopathy. A Golgi study

Generally accepted features of alcoholic fetopathy are delayed maturation and retarded dendritic development of neocortex, hippocampus and cerebellum. The present study investigates the effects of a membrane stabilizing agent (CDP-choline) on Purkinje cells of chronically alcohol intoxicated newborn rat pups, employing a Golgi impregnation technique. Both quantitative and qualitative data indicate that CDP-choline modifies the alcohol induced lesion.     

The Cleavage of Semaphorin 3C Induced by ADAMTS1 Promotes Cell Migration

Metastasis is a sequential process that allows cells to move from the primary tumor and grow elsewhere. Because of their ability to cleave a variety of extracellular signaling and adhesion molecules, metalloproteases have been long considered key components of the metastatic program. However, the function of certain metalloproteases, such as ADAMTS1, is not clear and seems to depend on the cellular environment and/or the stage of tumor progression. To characterize the function of ADAMTS1, we performed two alternative proteomic approaches, difference gel electrophoresis and stable isotope labeling by amino acids in cell culture, to identify novel substrates of the metalloprotease. Both techniques showed that overexpression of ADAMTS1 leads to the release of semaphorin 3C from the extracellular matrix. Although semaphorins are well known regulators of axon guidance, accumulating evidence shows that they may also participate in tumor progression. Here, we show that the cleavage of semaphorin 3C induced by ADAMTS1 promotes the migration of breast cancer cells, indicating that the co-expression of these molecules in tumors may contribute to the metastatic program.     

Evolution of marrow regeneration as revealed by transplantation studies

This study is concerned with the regeneration of bone marrow as an organized tissue. It is addressed specifically to the time in the regenerative program when the emerging tissue acquires the potential to reconstitute marrow. The evolution of this potential was investigated in rabbits by making autologous subcutaneous implants of tissue obtained at intervals after evacuation of a femur shaft. Analysis of 140 implant sites (89 of regenerating tissue and 51 of normal marrow) reveals a striking similarity in development of implants of a 2 to 4 day regenerating tissue and of normal marrow. New marrow encapsulated by bone can be seen in each instance 5 weeks after implantation. This property of regenerating tissue is uncovered before there are any obvious hemic elements. Significantly, the likelihood of a take is greatly increased when the implant contains a connective tissue. Marrow was always found in the implantation site when an ossicle was forming. It never occurred in the absence of bone. We conclude from this study that the appearance of modulated mesenchymal elements in the early regenerating tissue imparts the quality of normal marrow.     

The stepwise binding of small molecules to proteins. Nuclear magnetic resonance and temperature jump studies of the binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme.

The binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme was studied by both nuclear magnetic resonance (NMR) and temperature-jump methods under comparable conditions. The NMR measurements on the inhibitor spectrum were carried out over a range of inhibitor concentrations including levels at which most of the inhibitor was bound to the enzyme. Data in this region were obtained by a novel difference method in conjunction with correlation spectroscopy. The results from the combination of both experimental techniques demonstrated the existence of a two-step binding mechanism and produced both values for all of the individual rate constants and also the NMR spectral data for the inhibitor in the two enzyme-inhibitor complexes. The later data characterize the environment experienced by the inhibitor at each stage in the binding process and thus provides both a three-dimensional and a dynamic picture of the interaction.