Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGF1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGF1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGF1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGF1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFG1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B₁ retained their sensitivity to the effects of TGF1. Thus the loss of the inhibitory effect of TGF1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.Abbreviations TGF1 transforming growth factor 1 - BSA bovine serum albumin - FBS foetal bovine serum - BrdUrd bromodeoxyuridine - PI propidium iodide - PBS phosphate buffered saline     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Proteolytic specificity of chicken cathepsin L on bovineβ-casein

The proteolytic specificity of chicken cathepsin L was studied using bovine -casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P ₁ . Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P ₁ –P ₂ positions and that the S ₁ subsite can interact with modified amino acids such as phosphoserine.Abbreviations RP-HPLC reverse phase high performance liquid chromatography - NMec N-methyl coumarylamide - TEA triethylamine - TFA trifluoroacetic acid     

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.     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Partition coefficients of plant cuticles for monoterpenes

Summary Cuticle/water partition coefficients (K_c/w) for d-limonene, -pinene and -pinene were determined by an extrapolation and a desorption method. The sorption experiments were carried out with isolated angiosperm and gymnosperm cuticles and with [¹⁴C]-labelled monoterpenes, which were obtained biosynthetically. Both methods were suitable for the determination of the K_c/w of volatile hydrophobic compounds. For the angiosperm cuticles the partition coefficients are of the order of 10⁴, which indicates a high accumulation of monoterpenes in the cuticle. The values of the conifer cuticles of Picea abies (L.) Karst. and Abies alba Mill., however, are lower due to their high lignin content. This is proved by the increase of the partition coefficients after removal of polar and phenolic components. The K_c/w can be estimated with good accuracy from the octanol/water partition coefficient, which was determined experimentally.     

Dissimilatory hexaheme c nitrite reductase of “Spirillum” strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO _inf2 ^sup- × (mg protein × min)^-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/M_r 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.Abbreviations DNRA dissimilatory nitrate reduction to ammonia - EPR electron paramagnetic resonance - PAGE polyacrylamide gel electrophoresis - NaP_i sodium phosphate - SDS sodium dodecylsulfate