Gradient fractionation of cycling and resting cells monitored by BrdUrd incorporation

A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium Iodide (PI), showed the S compartment highly concentrated in the 1.073/77 g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

Cloning and characterization of the gene encoding an esterase from Spirulina platensis

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mauthner cell-initiated abrupt increase of the electric organ discharge in the weakly electric fishGymnotus carapo

Stimulation of the spinal cord of the electric fish Gymnotus carapo, evoked an abrupt increase in the discharge rate of the electric organ. At the maximum of this response, the rate increased an average of 26 ± 11.8%. The duration of the response was 4.9 ± 2.12 s; its latency was 10.4 ± 1.1 ms. Activation of the Mauthner axon played a decisive role in this phenomenon as indicated by the following: (1) recordings from the axon cap of the Mauthner cell demonstrated that the response was evoked if the Mauthner axon was antidromically activated and (2) a response that was similar to that produced by spinal cord stimulation, was elicited by intracellular stimulation of either Mauthner cell. Stimulation of the eighth nerve could also increase the discharge rate of the electric organ. The effect was greater if a Mauthner cell action potential was elicited. The findings described in the present report, indicate the existence of a functional connection between the Mauthner cell and the electromotor system in Gymnotus carapo. This connection may function to enhance the electrolocative sampling of the environment during Mauthner-cell mediated behaviors. This is a novel function for the Mauthner cell.Abbreviations EHP extrinsic hyperpolarizing potential - EOD electric organ discharge - M-AIR Mauthner initiated abrupt increase in rate - M-cell Mauthner cell - M-axon Mauthner axon - PM pacemaker nucleus - PM-cell pacemaker cell - PPn prepacemaker nucleus - SPPn sublemniscal prepacemaker nucleus     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.