Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

Interaction of Chlamydomonas dynein with tubulin

Studies were conducted to determine if dynein could bind to unpolymerized tubulin. Tubulin alone normally fractionated in the included volume of a molecular sieve Bio-Gel A-1.5m column. Incubated together, tubulin and dynein coeluted in the void volumn, suggesting that a complex had formed between the two. In addition, immunoelectron microscopy revealed preassembled microtubules were labeled with biotin antibody only when incubated in both dynein and biotinylated tubulin, evidence that dynein with bound biotinylated tubulin had decorated the microtubules. A fraction of the tubulin could be dissociated from dynein by addition of ATP and vanadate, as assayed by molecular sieve chromatography followed by densitometry of gels, suggesting that some tubulin bound to the B end of the dynein arm. Additional tubulin dissociated from the dynein under conditions of high salt. These studies, together with those indicating that tubulin blocked the A end of the dynein arm from binding to microtubules and promoted the interaction of two arms at their A ends, provide evidence that the A end of the arm also can bind tubulin. Thus, the tubulin subunits, themselves, on a microtubule rather than a particular surface lattice structure formed by adjacent protofilaments may provide the binding sites for both ends of the dynein arm.     

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.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

A hypothalamic activator of calmodulin-dependent enzymes is thymosin β4 (1–39)

A new class of stimulators of basal activity of a number of calmodulin-dependent enzymes have been previously isolated from bovine hypothalamus. One of these stimulators, denoted as C₃, has been purified to homogeneity by reverse phase HPLC and tentatively identified as thymosin ₄ (1–39) by mass spectrometry and Edman microsequence analysis. The stimulating effect of C₃ on rabbit skeletal muscle MLCK basal activity was compared with that of thymosin ₁ and thymosin ₄ (16–38). Evidence is presented that all the indicated compounds are Ca²⁺-independent high-affinity MLCK stimulators. The potency of the stimulators in activating the enzyme was: C₃>₄>(CaM+Ca²⁺>₁.This revised version was published online in June 2005 with corrections to the author name Gurvits.     

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

Mouse Chromosome 7

Chair of Committee for Mouse Chromosome 7     

Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains.

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine zeta 1-thrombin:human zeta 2-thrombin and human zeta 1-thrombin:bovine zeta 2-thrombin. Human zeta 1-thrombin and zeta 2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent zeta-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.