Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

The problem of natural antibodies, 1894–1905

As we have seen, natural antibodies first emerged as an experimental phenomenon without a plausible theoretical explanation. They were originally denied the status of antibody; then, adjustments to the side-chain theory transformed them from a curiosity into a foundation of the theory. However, in accommodating natural antibodies, Ehrlich had opened several holes in his mechanism of antibody formation.Thus, by 1905, natural antibodies were clearly established as problematic. From the practical standpoint, it seemed unwise to maintain an identity between normal and immune antibodies, given the therapeutic differences in their avidity. With the decline of Ehrlich's theory of antibody formation and the spread of Landsteiner's hapten technique for the production of antibodies against artificial antigens after World War I, the theoretical possibility of their existence as other than anomaly seemed more remote than ever. However, outside the theory and despite clinical considerations, natural antibodies remained a perplexing experimental phenomenon.⁴⁹     

Proteins and glycosaminoglycans in the intercellular matrix of the human cumulus-oophorus and their effect on conversion of proacrosin to acrosin.

Human cumuli-oophori were cultured in vitro in the presence of radioactive protein and polysaccharide precursors. The time course of the cumulus cell secretion was traced by histoautoradiography. Matrix solubilization, and sodium dodecyl sulphate polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that proteoglycan (Mr greater than 1,700,000) was the main cumulus cell product that was prevailingly deposited in the cumulus intercellular matrix and partly released into the culture medium. It was capable of accelerating the conversion of proacrosin to acrosin and this activity was abolished by enzymatic removal of chondroitin sulphate, the predominant glycosaminoglycan of this proteoglycan fraction. None of the other fractions, including a proteoglycan of Mr 80,000-90,000, containing heparan sulphate, accelerated the conversion of proacrosin to acrosin under the conditions used. The results suggest that chondroitin sulphate is the active component of the high-Mr proacrosin activator of the human cumulus-oophorus.     

Infraciliature, morphogenesis and life cycle of Endosphaera terebrans (Suctoria, Tokophridae)

The morphology, infraciliature, and life cycle of Endosphaera terebrans, a suctorian endocommensal of peritrichs, have been studied with the aid of silver impregnation. The life cycle of Endosphaera terebrans begins with infection of the host cell by a small larva. The swarmer has a pointed needle-like cellular projection and two rings of cilia. The swarmer penetrates the the peritrich, loses the cilia, and then matures into an adult. The infraciliature of the adult form has four rows of barren kinetosomes that lack kinetodesmal fibers. By endogenous budding, a migratory larva is produced that leaves the host cell through the peristomial disc and that can infect other peritrichs.     

Amplification of a Cryptosporidium parvum gene fragment encoding thymidylate synthase.

Currently, there is no effective therapy for cryptosporidiosis and it is unclear why antifolate drugs which are effective treatments for infections caused by closely related parasites are not also effective against Cryptosporidium parvum. In protozoa, the target of these drugs, dihydrofolate reductase (DHFR), exists as a bifunctional enzyme also manifesting thymidylate synthase (TS) activity and is encoded by a fused DHFR-TS gene. In order to prepare a probe to isolate the C. parvum DHFR-TS gene we have used degenerate oligonucleotides whose sequences are based on strongly conserved regions of TS protein sequence to prime the polymerase chain reaction (PCR) with C. parvum DNA. The PCR amplified a 375-bp DNA fragment which was cloned and sequenced; the deduced amino acid sequence had significant identity with known TS sequences, including strict conservation of all phylogenetically invariant TS amino acid residues. The cloned PCR fragment was used as a probe to isolate a number of overlapping clones from a C. parvum genomic library which were definitively shown to be of cryptosporidial origin by genomic Southern and molecular karyotype analyses. The deduced protein sequence of C. parvum TS was most similar to the bifunctional TS enzymes of Plasmodium chabaudi and Plasmodium falciparum.     

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.     

Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

Long-term low-dose mutation studies in human cells: metanil yellow and orange II

We tested the mutagenic effects of two commonly used fold colors, metanil yellow and orange II, in AHH-1 human lymphoblast cells. The cell line, which is competent for oxidative metabolism of various chemicals, was exposed to both compounds in high-dose x short-term (3 day) or high-dose x long-term (10-day) and low-dose x long-term (20-day) treatments. Concentrations of metanil yellow and orange II as low as 22 nM and 12 nM, respectively, were sufficient to induce mutation rates which were equal to twice the spontaneous mutation rate at the HPRT locus in AHH-1 cells.     

The effects of seed weight on growth and competitive ability of Rumex acetosella from two successional old-fields

Summary The effects of seed size on growth, biomass allocation and competitive ability of Rumex acetosella plants grown either individually or in competition were studied in two populations (6 months and 15 years old respectively) sampled from a postcultivation successional gradient. For plants grown individually there were highly significant effects of seed weight on growth after 43 days, with a higher relative growth rate (RGR) observed for plants raised from heavier seeds. However at the end of the experiment, seedlings developed from lighter seeds had a RGR 2 times greater than those from heavier seeds. Final biomass of the two types was not significantly different after 73 days of growth. When plants were grown individually, there were only slight differences between populations, but when grown in monocultures of 4 plants per pot, plants from the old population had higher root and leaf biomass per pot whereas those from the young population had a higher reproductive effort per pot. This suggests that a trade-off between allocation to sexual and vegetative reproduction occurs over successional time. In mixtures of light and heavy seeds, plants from light seeds were shorter, had fewer leaves and lower biomass than plants from heavy seeds, which were also taller and produced more dry matter than plants grown from heavy seeds in monoculture. The significant effects of seed weight and population on biomass parameters persisted unit the end of the experiment. Seedlings from heavy seeds were strong competitors: those from the young population grew better in the presence of neighbors than in monoculture and those from the late successional population suppressed the more the growth of their partners. Seedlings from light seeds were subordinate competitors. These results suggest that seedlings from seeds of different sizes benefit from contrasting ecological conditions and that selection acts on reproductive output along successional gradients.     

On the opportunity cost of the photosynthate invested in stem elongation reactions mediated by phytochrome

Summary Seedlings of shade-intolerant species react to alterations of the light climate caused by their neighbors with morphological changes that may influence the pattern of resource acquisition and utilization at the whole-canopy level. One such change, the increased stem elongation rate that is triggered by low red (R, 660 nm) to far-red (FR, 730 nm) ratios (R:FR) in dense canopies, might reduce the amount of assimilates available for leaf area expansion or root growth, and in that way affect resource capture by the canopy. We have tested this hypothesis by comparing the growth of both isolated individuals and canopies of the weed Amaranthus quitensis under conditions differing only in the spectral distribution of the incident light. When canopies received the full spectrum of sunlight, the stems were a large proportion (40–57%) of total biomass. Filtering the FR waveband (and hence raising the R:FR ratio to eliminate the neighbors' proximity-signal) resulted in shorter canopies with lighter stems. However, the growth of leaves and roots was not promoted by this treatment, indicating that the opportunity cost of the assimilates invested in the stems was nil or very small. Filtering the FR had no effect on biomass accumulation when plants were grown as isolated individuals. The higher growth of the canopics under full spectrum could be due to a higher light interception or to a higher efficiency of light conversion into biomass. The first possibility is weakened by the observation that filtering the FR had no effect on the dynamics of soil covering by the crops. The second is indirectly strengthened by results of an experiment with isolated plants showing that stem elongation, stem growth, and total plant biomass can be increased by reducing the flux of R light received by the stems without affecting the light climate of the leaves. Further work is needed to distinguish between these two possibilities; whatever the cause, our results show that the elongation responses to decreased R:FR may lead to a net increase in canopy productivity, and do not necessarily have a negative impact on the growth of resource-harvesting organs.