Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. Effects of mutations in the potential proteolytic cleavage site on processing and ligand binding.

The receptor for hepatocyte growth factor (HGF) is the product of the c-met proto-oncogene, a membrane-spanning tyrosine kinase receptor. To facilitate analysis of HGF and its receptor (HGFr), we expressed and purified a chimeric protein containing the extracellular domain (ECD) of the HGFr fused to the constant region of IgG heavy chain. This soluble form of the HGFr (sHGFr) bound HGF with an affinity similar to that of the authentic, membrane-associated receptor. The sHGFr also neutralized the binding of HGF to the HGFr expressed on A549 cells. Like the mature form of the HGFr, sHGFr is a heterodimer which arises by proteolytic processing within the ECD. In order to characterize the requirements for proteolytic processing of the ECD and the effects of cleavage on ligand binding, we expressed sHGFr variants containing amino acid substitutions in the putative processing site. Replacement of the P1 or P4 arginine, but not the P3 lysine, with alanine inhibited conversion to the alpha/beta heterodimer. This suggests that maturation is mediated by furin or a furin-like protease. Finally, we showed that processing of the sHGFr into the alpha/beta form is not required for high affinity binding to either pro- or mature HGF.     

Effects of progesterone and estradiol on the proliferation of phytohemagglutinin-stimulated human lymphocytes

In this paper we report on a study to elucidate whether the response of human lymphocytes to mitogenic stimulation was modified by physiological changes which occur during the menstrual cycle. Experiments with untreated cultures showed intra-individual variation to mitogen stimulation in female lymphocyte cultures, but a significant correlation between the menstrual cycle and the proliferation kinetics of lymphocytes was not found. Consequently, we performed experiments in which two of the hormones that regulate the menstrual cycle in women, estradiol and progesterone, were added to cultured human lymphocytes obtained from both men and women. The results indicate that both hormones at physiological concentrations have the capacity to modify the proliferation of PHA-stimulated human lymphocytes. Therefore, both hormones could play a role in the induction of the intra-individual variation observed in the untreated female cultures. However, in vivo other factors could also modify the proliferation kinetics of human lymphocytes preventing the demonstration of the effects of a single factor, such as the hormonal changes occurring during the menstrual cycle.     

Drought acclimation among tropical forest shrubs (Psychotria,Rubiaceae)

Summary Mechanisms of dry-season drought resistance were evaluated for five evergreen shrubs (Psychotria, Rubiaceae) which occur syntopically in tropical moist forest in central Panama. Rooting depths, leaf conductance, tissue osmotic potentials and elasticity, and the timing of leaf production were evaluated. From wet to dry season, tissue osmotic potentials declined and moduli of elasticity increased in four and five species, respectively. Irrigation only affected osmotic adjustment by P. furcata. The other seasonal changes in leaf tissue properties represented ontogenetic change. Nevertheless, they made an important contribution to dry-season turgor maintenance. Small between-year differences in dry season rainfall had large effects on plant water status. In 1986, 51 mm of rain fell between 1 January and 31 March, and pre-dawn turgor potentials averaged <0.1 MPa for all five Psychotria species in March (Wright 1991). In 1989, 111 mm of rain fell in the same period, pre-dawn turgor potentials averaged from 0.75 to 1.0 MPa for three of the species in April, and only P. chagrensis lost turgor. The relation between leaf production and drought differed among species. P. limonensis was buffered against drought by the lowest dry-season conductances and the deepest roots (averaging 244% deeper than its congeners) and was the only species to produce large numbers of leaves in the dry season. P. chagrensis was most susceptible to drought, and leaf production ceased as turgor loss developed. For the other species, water stress during severe dry seasons may select against dry-season leaf production.     

Comparative genet survival after fire in woody Mediterranean species

Summary Using data from three fires in northeastern Spain, we tested a condition necessary to support the idea that fire has been a factor in the evolution of the resprouting habit: populations of all resprouting species within a community should show high levels of genet survival after fires and show a low coefficient of variation. Species with high mean survival values were:Quercus ilex L.,Phillyrea latifolia L., andViburnum tinus L., with 88, 86 and 83% survival respectively; these groups had resprouts emerging from rootcrowns. Then followedArbutus unedo L. (75%),Pistacia lentiscus L. (73%),Erica arborea L. (77%),Erica multiflora L. (57%) andJuniperus oxycedrus L. (55%). This last group had resprouts from lignotubers or burls. These two groups also differed in the variability around the mean: the first showed a lower coefficient of variation, 6–12, and the second ranged from 19 to 26. Slope exposure had no significant influence on the process of resprouting, but soil depth did, with precipitation as a covariate. In the shallow soil category, the difference in genet survival between southern and northern exposures was 14% (71% vs. 57%); while the difference in the deep soil category was low, 5% (87% vs. 82%). There was no significant interaction. The component of variance for soils was larger than that for species-specific effects; substantial overlap of the within-species variance indicated that species responded as if they were a single hypothetical population, in which most of the variation in chances of survival was due to the soil conditions. The possession of the resprouting habit did not ensure a high performance. Hence, we find weak support for fire as a factor in the evolution of the resprouting habit.     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.     

Intrinsic Protein Phosphorylation in Synaptosomal Plasma Membrane Fragments: A Comparison of Cerebral Cortex Tissue from Several Species, Including Human Biopsy Specimens

Intrinsic protein phosphorylation was studied in synaptosomal membrane fragments made from cerebral cortex tissue taken from the following species: human (biopsy specimens), ox, rat, rabbit, guinea pig and mouse. Membrane fragments from all species exhibited a qualitatively similar range of protein acceptors phosphorylated by cyclic AMP-dependent protein kinase activity; contrary to a previous report, no evidence for cyclic GMP-dependent protein kinase activity was found in the human material. With the exception of membrane fragments prepared from ox brain, all the preparations exhibited the same range of Ca2+-dependent protein kinase activity. Ox brain obtained from a slaughterhouse yielded membranes containing no Ca2+-dependent protein kinase activity, but this may have been due to unavoidable postmortem losses.     

Epidermal growth factor initiates DNA synthesis after a time-dependent sequence of regulatory events in Swiss 3T3 cells—interactions with hormones and growth factors

Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14–15 hours. The final rate of initiation follows apparent first-order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4–6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases k, but after a delay of 4–6 hours. In contrast, both hydrocortisone and prostaglandin E₁ (PGE₁) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k. Prostaglandin F_2α (PGF_2α), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF_2α, nevertheless, act through different regulatory events.     

Stimulation of the initiation of DNA synthesis and cell division in several cultured mouse cell types : Effect of growth-promoting hormones and nutrients

The ability of prostaglandin F_2α (PGF_2α) and other prostaglandins to stimulate the initiation of DNA synthesis in quiescent cultures of various mouse fibroblastic cell types has been investigated. PGF_2α was found to be more effective than the other prostaglandins. Most cell types, with the exception of BALB/c 3T3, responded to PGF_2α. Addition of PGF_2α in combination with insulin resulted in a synergistic increase in the proportion of cells synthesizing DNA. The effect of nutrients on the stimulation of the initiation of DNA synthesis has been examined in detail; it was found that Swiss 3T3 cells showed a requirement for hypoxanthine and vitamin B₁₂ whereas Swiss 3T6 cells demonstrated a stringent requirement for vitamin B₁₂ only. The effect of prostaglandin precursors, synthetic analogues of the prostaglandin endoperoxides and inhibitors of prostaglandin synthesis was also examined in two cell types. The effect of PGF_2α was compared with that of two polypeptide growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF) in Swiss 3T6 cells grown in 0.0025% (v/v) serum. In combination with insulin each of these three growth factors stimulated the initiation of DNA synthesis in approximately the same number of cells.     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.