Contribution of heterotrophic plankton to nitrogen regeneration in the upwelling ecosystem of A Coruna (NW Spain)

The contribution of heterotrophic plankton to nitrogen (N) regenerationin the water column, and its significance for the requirementsof phytoplankton, were studied at the seasonal scale in thecoastal upwelling ecosystem of A Coruña (Galicia, NWSpain). During 1995–1997, monthly measurements were takenof hydrographic conditions, dissolved nutrients, and abundanceand biomass of microplanktonic heterotrophs (bacteria, flagellatesand ciliates), phytoplankton and mesozooplankton (>200 µm).Additionally, series of experiments were conducted to quantifyN fluxes, including primary production (¹⁴C method), phytoplanktonuptake of nitrate, ammonium and urea (¹⁵N-labelling techniques),microheterotrophic regeneration of ammonium, mesozooplanktongrazing (chlorophyll gut-content method) and excretion of ammoniumby mesozooplankton. Two N budgets were built for the averagesituations of high (>100 mg C m^-2 h^-1) and low (<100 mgC m^-2 h^-1) primary production. The results revealed that phytoplanktonrelied strongly on regenerated ammonium all year round (33 and43% of total N uptake in high and low production situations,respectively). This demand for ammonium was closely matchedby regeneration rates of microplankton (0.14–0.25 mmolN m^-2 h^-1), whereas zooplankton contributed on average <10%to N regeneration. Likewise, zooplankton grazing had littledirect control on phytoplanktonic biomass. The results obtainedindicate that in the A Coruña upwelling system, N biomassof heterotrophic plankton is generally higher than phytoplanktonN biomass. The high rates of N regeneration measured also suggestthat a large proportion of the organic matter produced afteran upwelling pulse is recycled in the water column through themicrobial food web.     

Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.     

Laser-based micromanipulation for separation and identification of individual Frankia vesicles

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.     

Condensation of DNA and chromatin by histones H1, H5 and protamines. The influence of a protein phosphorylation

A mutual displacement of the DNA crosslinking protiens, protamines, histones H1 and H5, is observed if cell nuclei are incubated in solutions containing one of these species. While their competition is in accord with their specific positive charge, a displacement of the core histones does not occur. Crosslinking proteins give rise to the formation of macrocomplexes from DNA and core particles, respectively which in the present study are investigated by stray-light and centrifugation methods. It is shown than the reduction of electrostatic affinities, which in vivo is achieved by a protein phosphorylation, serves to enhance complex sizes, probably by permitting at thermodynamically controlled, ordered association of DNA segments.     

An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.

Viruses used for gene therapy are usually genetically modified to deliver therapeutic transgenes and prevent viral replication. In contrast, replication-competent viruses may be used for cancer therapy because replication of some viruses within cancer cells can result in their destruction (oncolysis). Viral ribonucleotide reductase expression is defective in the HSV1 mutant hrR3. Cellular ribonucleotide reductase, which is scarce in normal liver and abundant in liver metastases, can substitute for its viral counterpart to allow hrR3 replication in infected cells. Two or three log orders more of hrR3 virions are produced from infection of colon carcinoma cells than from infection of normal hepatocytes in viral replication assays. This viral replication is oncolytic. A single intravascular administration of hrR3 into immune-competent mice bearing diffuse liver metastases dramatically reduces tumor burden. hrR3-mediated tumor inhibition is equivalent in immune-competent and immune-incompetent mice, suggesting that viral oncolysis and not the host immune response is the primary mechanism of tumor destruction. HSV1-mediated oncolysis of diffuse liver metastases is effective in mice preimmunized against HSV1. These results indicate that replication-competent HSV1 mutants hold significant promise as cancer therapeutic agents. Yoon, S. S., Nakamura, H., Carroll, N. M., Bode, B. P., Chiocca, E. A., Tanabe, K. K. An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata. III. The shaping of the body column

Excised pieces of hydra body tissue of varying size and shape regenerate into cylinders with a head and foot at opposite ends. The numbers of cells along the axial and circumferential dimensions were determined before, during, and after regeneration. The main process in shaping the excised tissue into a body column was found to be a rearrangement of the cells. When regenerates of different size were measured, the proportions of the body columns were found to vary, such that the smaller the animal the squatter the body column was. The presence of the head in regenerates was necessary for the formation or maintenance of the cylindrical shape, while the size of the head determined the proportions of the cylinder. The formation of a gradient of adhesivity induced by the developing head is suggested as the basis for the rearrangement of the cells into the cylindrical form.     

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.     

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 +/- 30 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.     

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.