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.     

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.     

中国西北沙漠夜间活动的东鳖甲属昆虫二新种(鞘翅目:拟步甲科:漠甲亚科)

记述中国西北地区东鳖甲属2新种:巴丹东鳖甲A.badainica Ba,Ren&Liu sp.nov.和古尔班东鳖甲A.gurbantunggutica Ba&Ren sp.nov.,提供了主要鉴别特征和形态图,并简要讨论了其昼夜活动规律.     

Haemosporidian Parasites of Antelopes and Other Vertebrates from Gabon,Central Africa

Re-examination, using molecular tools, of the diversity of haemosporidian parasites (among which the agents of human malaria are the best known) has generally led to rearrangements of traditional classifications. In this study, we explored the diversity of haemosporidian parasites infecting vertebrate species (particularly mammals, birds and reptiles) living in the forests of Gabon (Central Africa), by analyzing a collection of 492 bushmeat samples. We found that samples from five mammalian species (four duiker and one pangolin species), one bird and one turtle species were infected by haemosporidian parasites. In duikers (from which most of the infected specimens were obtained), we demonstrated the existence of at least two distinct parasite lineages related to Polychromophilus species (i.e., bat haemosporidian parasites) and to sauropsid Plasmodium (from birds and lizards). Molecular screening of sylvatic mosquitoes captured during a longitudinal survey revealed the presence of these haemosporidian parasite lineages also in several Anopheles species, suggesting a potential role in their transmission. Our results show that, differently from what was previously thought, several independent clades of haemosporidian parasites (family Plasmodiidae) infect mammals and are transmitted by anopheline mosquitoes.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

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.