Purification and characterization of recombinant human interleukin 4. Biological activities, receptor binding and the generation of monoclonal antibodies.

A synthetic gene coding for human interleukin 4 (IL-4) was cloned and expressed in Saccharomyces cerevisiae (baker's yeast) as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence, resulting in secretion of mature IL-4 into the culture medium (0.6-0.8 micrograms/ml). A protocol was developed for purification of this protein. Crude cell-free conditioned medium was passed over a concanavalin A-Sepharose affinity column; bound proteins were eluted and further purified by S-Sepharose Fast Flow cation exchange and C18 reverse-phase h.p.l.c. Highly purified IL-4 was obtained by this method (0.3-0.4 mg per litre of culture) with a recovery of 51%. Thermospray liquid chromatography-mass spectrometry showed the C-terminal N-glycosylation site to be largely unmodified, and also showed that the N-terminus of the purified recombinant IL-4 (rIL-4) was authentic. Thiol titration revealed no free cysteine residues, implying that there are three disulphide groups, the positions of which remain to be determined. We have characterized the biological activities of the purified rIL-4. This material is active in B-cell co-stimulator assays, T-cell proliferation assays and in the induction of cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells. Half-maximal biological activity of the rIL-4 was achieved at a concentration of 120 pM. We have radioiodinated rIL-4 without loss of biological activity and performed equilibrium binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding site with high affinity (Kd = 100 pM) and revealed 1100 receptors per cell. Receptor-ligand cross-linking studies demonstrated a single cell-surface receptor with an apparent molecular mass of 124 kDa. Two monoclonal antibodies have been raised to the human rIL-4, one of which blocks both the biological activity of rIL-4 and binding to its receptor.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

Centrosome reorientation in regenerating endothelial monolayers requires bFGF

Monolayers of endothelial cells respond to physical denudation with a characteristic sequence of lamellipodia extrusion, cell migration, and cell proliferation. Basic fibroblast growth factor (bFGF) has been implicated as a necessary component of this process: addition of exogenous bFGF enhances monolayer regeneration both in vitro and in vivo, and monolayer regeneration can be inhibited in vitro by treatment with neutralizing antibodies raised against bFGF. Centrosome reorientation from a random location to one preferentially situated between the nucleus and the denudation edge has been postulated as a mechanism essential for cell polarization and subsequent migration. This present study examined the effects of a polyclonal antibody to bFGF and suramin on monolayer regeneration, actin microfilament staining, and centrosome orientation at the wound edge of partially denuded bovine large vessel endothelial monolayers. Treatment with anti-bFGF or suramin abolished monolayer repair in these cultures. Cells at the denudation edge showed altered actin staining patterns and reduced lamellipodia extrusion, and there was complete inhibition of centrosome reorientation in treated cultures. Monolayer repair and centrosome reorientation could be restored by addition of exogenous bFGF in antibody but not suramin treated cultures. Recent evidence suggests that preferential centrosome location in migrating cells may be a consequence of lamellipodia protrusion and cell spreading, rather than an indication of cell polarization. However, these results indicate that agents which interfere with bFGF availability prevent endothelial monolayer regeneration via mechanisms involving cell spreading and/or centrosome reorientation.     

Le ionophore A23187

The acrosomic status of spermatozoa prepared for IVF has been evaluated by means of immunofluorescence test from Fenichel and Hsi using calcium A 23187 ionophore as inductor of acrosome reaction (AR). The spontaneous AR remains slight, even after 6 hour-incubation in Menezo B2 (6,8+2,7%). The response to ionophore, moderate before (11,2+9%), frankly increases after a 6h-capacitation (28,9+8,3%) in a group of 25 IVF couples (tubal indication, normal semen, positive fertilization). Nevertheless, it remains slight or null in 4 cases of unexplained repeated failure of fertilization. The response to ionophore A 23187 allows to explore the kinetics of capacitation of spermatozoa and their ability to perform AR. Its significance in terms of fecondance remains to be precised.     

Behavioural and chemical mechanisms behind a Mediterranean ant–ant association

1. Interspecific competition among ants is common, and so is competitive exclusion among dominant ant species. In contrast, specific associations between non‐parasitic ant species are rare, especially in the temperate zones. As an exception, the subordinate ant Camponotus lateralis frequently co‐occurs with the dominant Crematogaster scutellaris but rarely with other dominant ants. 2. This association is one of various associations between Camponotus and Crematogaster species across the world. However, the mechanisms behind these co‐occurences are largely unknown. 3. In the present study, we therefore investigated the association of Ca. lateralis and Cr. scutellaris. We studied the spatial association of the nests, interspecific aggression, both species' cuticular hydrocarbon profiles, and their propensity to follow the other species' pheromone trails. 4. Crematogaster scutellaris usually attacked and displaced the generally submissive Ca. lateralis, but was significantly less aggressive at jointly used trails. Camponotus nests were always in close proximity to Crematogaster nests. 5. The cuticular hydrocarbons of both species consisted of alkanes with chain lengths between C21 and C35. The two species had 25 hydrocarbons in common, including mono‐, di‐, and tetramethyl alkanes. Despite this qualitative similarity, however, the quantitative hydrocarbon composition differed between the two species. 6. Camponotus lateralis followed artificial trails containing trail pheromones of Cr. scutellaris, but the latter did not follow Ca. lateralis trail pheromones. Interspecific trail‐following by Camponotus, but not vice versa, has been observed in another Camponotus–Crematogaster association and may be a more general mechanism that facilitates associations between the two ant genera.     

Effects of relaxin on matrix remodeling enzyme activity of cultured equine ovarian stromal cells

Relaxin participates in extracellular matrix (ECM) remodeling in many reproductive organs, including the ovary, by regulating proteolytic enzyme activity. Accumulated evidence indicates this action of relaxin is involved in ovarian follicle development and ovulation. Equine follicles are embedded in cortex that is at the center of the ovary and they must expand/emigrate to the fossa, the only site in the ovary for ovulation. Due to the tremendous expansion of the follicle in this species, we hypothesized that ovarian stromal remodeling would be extensive. Therefore, cultured equine ovarian stromal cell (EOSC) lines were obtained from stroma at the apex of large follicles and the effects of relaxin on gelatinases A and B, tissue inhibitors of matrix metalloproteinases (TIMPs), plasminogen activators (PAs) and PA inhibitor-1 (PAI-1) activities were assessed. Our results showed that equine relaxin increased the activity of total gelatinase A (both pro forms and mature forms) and latent progelatinase B present in conditioned medium, latent progelatinase A present in cell extracts, and TIMP-1 and TIMP-2 present in conditioned medium. This study also revealed that equine relaxin increased the urokinase-type PA activity in conditioned medium and cell extracts, tissue-type PA activity in ECM and PAI-1 activity in conditioned medium. These results suggest that relaxin may contribute to equine follicle growth and migration, and facilitate ovulation by modulating the degradation of ECM in ovarian stromal tissue.     

A paradigm for therapy-induced microenvironmental changes in solid tumors leading to drug resistance

Intrinsic alterations in the tumor microenvironment are known to contribute to various forms of drug resistance. For example, tumor hypoxia, due to abnormal or sluggish blood flow within areas of solid tumors, can result in both microenvironment-mediated radiation and chemotherapeutic drug resistance. In contrast, acquired resistance to chemotherapy is generally considered to be the result of the gradual selection of mutant subpopulations having genetic mutations and biochemical alterations responsible for the resistant phenotype. Here we present a paradigm for therapyinduced microenvironment-mediated acquired drug resistance. It is based on the results showing that tumor cells appear to be heterogeneous in their relative dependence on adjacent tumor-associated vasculature for survival. Some tumor cells are highly vessel dependent, whereas some are significantly less so, and thus can survive in more hypoxic regions of tumors, distal from such tumor vessels. Hence, it is possible that variant tumor cells that are less vessel dependent may therefore be selected for over time by successful antiangiogenic drug therapies. This results in loss of response or attenuated responses to the therapy. Preliminary evidence is summarized in support of this hypothesis, using paired human colon cancer (HCT116) cell lines that contain two copies of either the wild-type or the disrupted p53 tumor suppressor gene. The mutant cells were found to be less responsive to antiangiogenic therapy, compared to the wild-type cells, and could be progressively selected for in mixed cell populations. Because p53 inactivation can lead to resistance to hypoxia-mediated apoptosis, the results suggest that a protracted and successful antiangiogenic therapy may create more hypoxic tumor microenvironments, thereby creating the necessary conditions to accelerate the selection of mutant tumor cells that are more adept in surviving and growing in such environments. As such, consideration might be given to the combined use of bioreductive hypoxic cell cytotoxic drugs and angiogenesis inhibitors to prolong the efficacy of antiangiogenic therapeutics.     

The generation and characterization of antagonist RNA aptamers to human oncostatin M

Oncostatin M (OSM) is a multifunctional member of the interleukin-6 cytokine family. OSM has been implicated as a powerful proinflammatory mediator and may represent a potentially important, novel therapeutic opportunity for treatment of established rheumatoid arthritis. To further investigate the role of OSM in inflammatory disorders, we have isolated a series of RNA aptamers that bind specifically to human OSM. The highest affinity aptamer, designated ADR58, has been characterized in a series of in vitro and cell based assays. ADR58 has an affinity of 7 nm for human OSM, and it can antagonize OSM binding to the gp130 receptor and specifically antagonize OSM mediated signaling. The aptamer has been truncated in length to 33 bases, all pyrimidine positions are substituted with 2' fluorine, and 14 of 18 purine positions have been substituted with 2' O-methyl to increase stability toward nucleases. This truncated, modified form of ADR58 retains complete affinity and functional activity for OSM. This aptamer may be used as a tool to further investigate the role of OSM in inflammatory disorders and may also have role as a therapeutic agent.