Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Salicyloylaspartate as an endogenous component in the leaves of Phaseolus vulgaris

An amide conjugate of o-methoxybenzoic acid and aspartic acid has been isolated from bean leaves. After extraction and methylation of plant material, this compound was isolated as two isomeric monoethyl monomethyl esters. The ethylation of the aspartyl carboxyl groups was shown to be a likely result of an extraction procedure utilising acidified ethanol, the methylation of the aromatic hydroxy of the methoxy group to be due to the derivatisation procedure. Studies with pentafluorobenzylation confirmed that the endogenous compound is o-hydroxybenzoylaspartate.     

Sexual dimorphism in the early Miocene species ofProconsul from the Kisingiri Formation of east Africa: A morphometric examination using multivariate statistics

This paper examines the pattern(s) of sexual dimorphism within the upper dentition ofProconsul specimens from the early Miocene of east Africa. These fossils are compared against the corresponding dentition ofPan troglodytes andGorilla gorilla using principal components and cluster analyses. This paper demonstrates that both sexes ofPan andGorilla are characterized by their own distinctive shape patterns. It is also demonstrated that someProconsul specimens examined here display a pattern that is dissimilar from otherProconsul specimens also examined. This suggests that at least two species ofProconsul may have to be recognized as having lived in this region during the early Miocene. The identification of distinct patterns withinProconsul also suggests that their overall shape and size range are more similar toPan than toGorilla.     

Influence of mefluidide on growth,development, and cell wall digestibility of sorghum

Application of mefluidide (N-[2,4-dimethyl-5-([(trifluoromethyl)sulfonyl]amino) phenyl]acetamide) inhibits plant development in perennial grasses. This study examined the effect of mefluidide on the morphological development and digestibility of sorghum. In the greenhouse, 5.9 × 10^–5 g active ingredient (a.i.) plant^–1 applied at the seedling, eight-leaf and boot stages reduced mean plant height 70%, 59%, and 2%, respectively. Heights were also reduced 14%, 15% and 35% by 5.9 × 10^–8, 5.9 × 10^–7 and 5.9 × 10^–6 gram a.i. plant^–1 applied at the eight-leaf stage. Field application of 0.26 or 0.52 kg ha^–1 mefluidide at either the eight-leaf or flagleaf stage reduced mean plant height of all cultivars. Basal tiller numbers increased 319% 28 d, and dry matter production was reduced 65% 42 d following mefluidide application at the eight-leaf stage. Treated stems were 34% higher and treated leaves were 7% higher in cellulase dry matter digestibility than control plants following mefluidide application at the eight-leaf stage. These results indicate that mefluidide application to vegetative stages in sorghum may enhance the forage value of the plants while it inhibits normal plant growth.     

Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis

Whereas capsulate strains of Neisseria meningitidis are dependent on pili for adhesion to human endothelial and epithelial cells, strains which lacked assembled pili and were partially capsule-deficient adhered to and invaded human endothelial and epithelial cells if they expressed the Opc protein. Bacteria expressing low or undetectable levels of Opc protein failed to adhere to or invade eukaryotic cells. In addition, the presence of OpaAC751 protein on the surface of bacteria did not increase bacterial interactions with host cells. Association of Opc-expressing bacteria was inhibited by antibodies against Opc. Invasion was dependent on the host-cell cytoskeletal activity and was inhibited by cytochalasin D. In some cells, infected at the apical surface, bacteria emerging from basal surface were detected by electron microscopy. Opc is found in diverse meningococci and may represent a common virulence factor which facilitates adherence and invasion by these bacteria.     

Enzyme activity in organic solvent as a function of water activity determined by membrane inlet mass spectrometry

Summary Membrane inlet mass spectrometry (MIMS) is introduced as a method for measuring water activity in nonpolar solvents, aqueous solutions and gas phase. The determination of the rate of hydrolysis of diphenyl carbonate by porcine liver esterase as a function of water activity in diisopropyl ether is presented as an example. A linear relationship is found between the enzyme activity and the water activity.     

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.     

Human and mouse S-protein mRNA detected in Northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis

Summary Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.