Interferon inhibits DNA Synthesis induced in Mouse Lymphocyte Suspensions by Phytohaemagglutinin or by Allogeneic Cells

IN addition to its well known antiviral activity, interferon has recently been shown to inhibit the multiplication of tumour and mammalian cells in cell culture^1–6. We report here the inhibition by interferon of DNA synthesis induced in mouse spleen lymphocytes by the non-viral stimuli phytohaemagglutinin (PHA) and allogeneic lymphocytes. These findings are in accord with our contention that interferon affects cell function and, furthermore, they suggest that by acting on lymphocytes, interferon plays a role in the immunological response of the host.     

Changes in the levels of wheat- and barley-germ agglutinin during embryogenesis in vivo,in vitro and during germination

Radioimmunoassay has been used to measure levels of wheat-germ agglutinin and barley-germ agglutinin during embryogenesis and germination. The two lectins exhibited similar patterns of accumulation during grain maturation in vivo and both decreased to low levels after imbibition of harvest-ripe grains for 3 d. Precocious germination of immature wheat and barley embryos excised and cultured in vitro could be prevented either by inclusion of abscisic acid or mannitol in the culture medium. Changes in the level of wheat-germ agglutinin induced by in vitro culture depended on the maturation stage of the embryo. No direct correlation was found between application of exogenous abscisic acid and accumulation of the lectin.     

Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis

A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105. This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments. Two of the new vectors phi 105J27 and phi 105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.     

Virus protein changes and RNA termini alterations evolving during persistent infection

Cloned infectious vesicular stomatitis virus isolated following 5 years of persistent infection of BHK21 cells in vitro exhibits a number of peptide map changes in the G protein (spike glycoprotein), the M protein (membrane matrix protein) and the N protein (nucleocapsid structural protein). Only slight alterations have occurred in the peptide maps of the two VSV polymerase-associated proteins L and NS. Dideoxy sequencing of the 3′ ends of the cloned virus originally used to establish the persistent infection, and of the cloned virus recovered following 5 years of persistence, shows one base substitution in the three base junction between the 3′ leader sequence and the N protein-coding region. Repeated lytic passages of virus recovered from persistent infection led to no oligonucleotide map changes after 30 passages, but two map changes were present after 102 and remained after 133 lytic passages in BHK21 cells in vitro. Only one of these represented reversion to the original map position, and this “mutant” virus still exhibited a temperature-sensitive small plaque phenotype. Finally, the mutated virus recovered after more than $\text{5}\text{1}\text{2}$ years of persistent infection is now so slow-growing that it can establish persistent infection of BHK21 cells in the absence of DI particles (although DI particles are present constantly once the cells recover from the initial cytopathology).     

Quantitative determination of the induction of apoptosis in a murine B cell line using flow cytometric bivariate cell cycle analysis.

WEHI-231 cells have been used extensively as a model of tolerance induction in B cells. Recent evidence has shown that anti-IgM treatment of WEHI-231 cells resulted in the induction of apoptosis. In this study, using acridine orange staining and flow cytometric analysis, we demonstrated that apoptotic cells are detected as a distinct population of cells separate from the cells in normal cell cycle progression. The validity of analysis gates was confirmed by cell sorting of the apoptotic population versus normal cells and subsequent gel analysis. Using this technique, we have demonstrated that F(ab')2 anti-mu, A23187, or PMA induced apoptosis in the WEHI-231 cells. The addition of LPS reversed apoptotic induction as seen previously with the WEHI-231 cell line. In contrast, however, PMA did not prevent the induction of apoptosis in anti-mu-treated cells. Additionally, we were interested in determining if the induction of apoptosis was restricted to a specific phase of cell cycle. Since growth inhibition results in most cells arresting in the G1 phase of cell cycle, we wanted to demonstrate apoptosis as a G1-dependent event. This was examined with WEHI-231 cells treated with known cell cycle inhibitors. Interestingly, inhibition of cells in each phase of cycle resulted in the induction of apoptosis. LPS was able to inhibit the induction of apoptosis with each of the cell cycle inhibitors except actinomycin D. Furthermore, we have demonstrated that the WEHI-231 cells contain a Ca(2+)-Mg(2+)-dependent preexisting endonuclease.     

Two Amino-acid Carriers in Pea Chloroplasts

CHLOROPLASTS are more permeable to amino-acids^1,2 than are cell membranes³ and also discriminate between their optical isomers². This has led to the suggestion that the passage of amino-acids through the limiting membranes of chloroplasts is facilitated by carriers². Using the additional criteria of saturation and competition, we support this suggestion and show that at least two carriers are probably involved.     

A predictive model for freshwater bioassessment (Mondego River,Portugal)

We sampled macroinvertebrates at 75 locations in the Mondego river catchment, Central Portugal, and developed a predictive model for water quality assessment of this basin, based on the Reference Condition Approach. Sampling was done from June to September 2001. Fifty-five sites were identified as “Reference sites” and 20 sites were used as “Test sites” to test the model. At each site we also measured 40 habitat variables to characterize water physics and chemistry, habitat type, land use, stream hydrology and geographic location. Macroinvertebrates were generally identified to species or genus level; a total of 207 taxa were found. By Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering and analysis of species contribution to similarities percentage (SIMPER), two groups of reference sites were established. Using Discriminant Analysis (stepwise forward), four variables correctly predicted 78% of the reference sites to the appropriate group: stream order, pool quality, substrate quality and current velocity. Test sites’ environmental quality was established from their relative distance to reference sites, in MDS ordination space, using a series of bands (BEAST methodology). The model performed well at upstream sites, but at downstream sites it was compromised by the lack of reference sites. As with the English RIVPACS predictive model, the Mondego model should be continually improved with the addition of new reference sites. The adaptation of the Mondego model methodology to the Water Framework Directive is possible and would consist mainly of the integration of the WFD typology and increasing the number of ellipses that define quality bands. Handling editor: K. Martens