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Query: UNIPROT:P06889 (Mol)
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The three-dimensional crystal structure of bovine trypsinogen at approximately pH 7.5 was initially solved at 2.6 A resolution using the multiple isomorphous replacement method. Preliminary refinement cycles of the atomic coordinates trypsinogen have been carried out first to a resolution of 2.1 A, and later to 1.9 A, using constrained difference Fourier refinement; During the process, structure factors Fc and phi c were calculated from the trypsinogen structure and final interpretation was based on an electron-density map computed with terms (2 Fo - Fc) and phases phic at a resolution of 1.9 A. Crystals of trypsinogen grown from ethanol-water mixtures are trigonal with space group P3121, and cell dimension a = 55.17 A and c = 109.25 A. The structure is compared with the bovine diisopropylphosphoryltrypsin structure at approximately pH 7.2, oirginally determined from orthohombic crystals by Stroud et al. (Stroud, R.M., Kay L.M., and Dickerson, R.E. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 125-140; Stroud, R.M., Kay, L.M., and Dickerson, R.E. (1974), J. Mol. Biol. 83, 185-208), and later refined at 1.5 A resolution by Chambers and Stroud (Chambers, J.L., and Stroud, R.M. (1976), Acta Crystallogr. (in press)). At lower pH, 4.0-5.5 diogen, with cell dimensions a = 55.05 A and c = 109.45 A. This finding was used in the solution of the six trypsinogen heavy-atom derivatives prior to isomorphous phase analysis, and as a further basis of comparison between trypsinogen and the low pH trypsin structure. There are small differences between the two diisopropylphosphoryltrypsin structures. Bovine trypsinogen has a large and accessible cavity at the site where the native enzyme binds specific side chains of a substrate. The conformation and stability of the binding site differ from that found in trypsin at approximately pH 7.5, and from that in the low pH form of diisopropylphosphoryltrypsin. The catalytic site containing Asp-102, His-57, and Ser-195 is similar to that found in trypsin and contains a similar hydrogen-bounded network. The carboxyl group of Asp-194, which is salt bridged to the amino terminal of Ile-16 in native trypsin or other serine proteases, is apparently hydrogen bonded to internal solvent molecules in a loosely organized part of the zymogen structure. The unusually charged N-terminal hexapeptide of trypsinogen, whose removal leads to activation of the zymogen, lies on the outside surface of the molecule. There are significant structural changes which accompany activation in neighboring regions, which include residues 142-152, 215-550, 188A-195. The NH group of Gly-193, normally involved in stabilization of reaction intermediates (Steitz, T.A., Henderson, R., and Blow, D.M. (1969), J. Mol. Biol. 46, 337-348; Henderson, R. (1970), J. Mol. Biol. 54, 341-354; robertus, J.D., Kraut, J., Alden, R.A., and Birkoft, J.J. (1972), Biochemistry 11, 4293-4303) in the enzyme, is moved 1.9 A away from its position in trypsin...
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PMID:Structure of bovine trypsinogen at 1.9 A resolution. 55 51

An improved 2.5-A electron density map of chymotrypsinogen was calculated by incorporating heavy-atom anomalous scattering effects and a new model of the molecule was constructed. Phases from x-ray structure factors (R = 0.43) computed from this model were then used in the calculation of another electron density map against which the model was further refined. The catalytic Ser-195 side chain in the new model is in the "down" or "acyl" orientation and its Ogamma atom is in position to form a normal hydrogen bond with Nepsilon2 of His-57. In contrast, the corresponding hydrogen bond in alpha-chymotrypsin (Birktoft, J.J., and Blow, D.M. (1972), J.Mol. Biol. 68, 187) is severely distorted, probably as a consequence of a 1.5-A shift in the relative positions of the two cylindrical folding domains composing most of the molecule. We suggest that this activiation induced distortion of the charge-relay, hydrogen-bonding system plays an important role in the genesis of enzymic activity, in accord with an earlier proposal by Wang concerning the role of bent hydrogen bonds in enzyme catalysis (Wang, J.J. (1970), Proc. Natl. Acad. Sci. U.S.A. 66, 874).
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PMID:A detailed structural comparison between the charge relay system in chymotrypsinogen and in alpha-chymotrypsin. 97 71

Leaf thionins of several barley cultivars and wild barley species were analysed. We found large differences in the numbers of leaf thionin genes in different Hordeum species. While, for instance, cultivars of Hordeum vulgare (Section Hordeum) contain more than 50 copies of thionin genes per haploid genome, the numbers are much lower in Hordeum species belonging to the sections Critesion and Stenostachys. The apparent number of genes correlates with the concentration of leaf thionin and its mRNA, which differs more than 100-fold among various Hordeum species. Leaf thionins are synthesized as high molecular weight precursor proteins that contain a signal peptide domain, a thionin domain and an acidic polypeptide domain. Analysis of cDNA clones of leaf thionins revealed a family of related transcripts. When the predicted amino acid sequences of the precursor molecules of wild barley species were compared, differences in the sequence variability of the three domains became apparent. The frequency of amino acid exchanges is much higher within the thionin domain than in the signal peptide and acidic polypeptide domains. The amino acid exchanges within the thionin domain do not occur at random but are confined to variable regions that alternate with highly conserved areas. Conserved regions comprise mostly cysteine residues and adjacent amino acids and may be important for the correct formation of the specific disulphide configuration of thionins.
Mol Gen Genet 1992 Feb
PMID:A comparison of leaf thionin sequences of barley cultivars and wild barley species. 137 80

Dust mite allergens are considered as a major cause of allergic disease and as a risk factor for asthma. Der p I, a 222 amino-acid residue globular glycoprotein, is one of the major allergens from Dermatophagoides pteronyssinus (Dpt) mites. In this study, we have used predictive conventional algorithms (i.e. hydrophilicity, mobility, accessibility) and a three-dimensional model of Der p I derived from comparison to actinidin and papain to select continuous amino acid sequences as potential B cell epitopes. Four peptides, 52-71, 117-133, 176-187, 188-199 were synthesized. Their antigenic reactivity was investigated, mainly by measuring their capacity to induce in vitro histamine release. Results indicated that only Dpt-sensitive patients react specifically to Der p I-derived peptides and more frequently to 52-71 and 117-133. For each peptide, the intensity of response was dependent on the patient tested and on the peptide concn. The capacity of peptides to induce histamine release was demonstrated to be correlated with the serum level of anti-Der p I IgE (r = 0.86; p less than 10(-2)). Taken together these data emphasize, in Dpt-sensitive patients, the heterogeneity of the specific response to synthetic Der p I-derived peptides and underline the possible variety of epitopes belonging to the allergen Der p I.
Mol Immunol 1992 Jun
PMID:Specific histamine release capacity of peptides selected from the modelized Der p I protein, a major allergen of Dermatophagoides pteronyssinus. 137 13

Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.
Plant Mol Biol 1992 Dec
PMID:Factors influencing Agrobacterium-mediated transient expression of gusA in rice. 146 39

Members of the Myc family of proteins share a number of protein motifs that are found in regulators of gene transcription. Conserved stretches of amino acids found in the N-terminal transcriptional activation domain of c-Myc are required for cotransforming activity. Most of the Myc proteins contain the basic helix-loop-helix zipper (bHLH-Zip) DNA-binding motif which is also required for the cotransforming activity of c-Myc. L-Myc, the product of a myc family gene that is highly amplified in many human lung carcinomas, was found to cotransform primary rat embryo cells with an activated ras gene. However, L-Myc cotransforming activity was only 1 to 10% of that of c-Myc (M. J. Birrer, S. Segal, J. S. DeGreve, F. Kaye, E. A. Sausville, and J. D. Minna, Mol. Cell. Biol. 8:2668-2673, 1988). We sought to determine whether functional differences between c-Myc and L-Myc in either the N-terminal or the C-terminal domain could account for the relatively diminished L-Myc cotransforming activity. Although the N-terminal domain of L-Myc could activate transcription when fused to the yeast GAL4 DNA-binding domain, the activity was only 5% of that of a comparable c-Myc domain. We next determined that the interaction of the C-terminal bHLH-Zip region of L-Myc or c-Myc with that of a Myc partner protein, Max, was equivalent in transfected cells. A Max expression vector was found to augment the cotransforming activity of L-Myc as well as that of c-Myc. In addition, a bacterially synthesized DNA-binding domain of L-Myc, like that o c-Myc, heterodimerizes with purified Max protein to bind the core DNA sequence CACGTG. To determine the region of L-Myc responsible for its relatively diminished cotransforming activity, we constructed chimeras containing exons 2 (constituting activation domains) and 3 (constituting DNA-binding domains) of c-Myc fused to those of L-Myc. The cotransforming potencies of these chimeras were compared with those of full-length L-Myc of c-Myc in rat embryo cells. The relative cotransforming activities suggest that the potencies of the activation domains determine the cotransforming efficiencies for c-Myc and L-Myc. This correlation supports the hypothesis that the Myc proteins function in neoplastic cotransformation as transcription factors.
Mol Cell Biol 1992 Jul
PMID:Activation domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells. 162 Jan 20

Forty-three patients with disseminated refractory malignancies each received an individually specified combination of either Adriamycin (n = 24) or mitomycin-C (n = 19) conjugated to a cocktail of murine monoclonal antibodies (mAb). Cancers were typed with both immunohistochemistry and flow cytometry using a panel of antibodies. Cocktails of up to six antibodies were selected based on total binding of greater than 80% of the malignant cells in the biopsy specimen. These mAb cocktails were then drug conjugated, safety tested, and administered intravenously. The Adriamycin immunoconjugates were well tolerated in 22/24 patients, with 17/24 having significant side effects. Fever, chills, pruritus, and skin rash were by far the most common transitory reactions. All were well controlled with premedication. A total of up to 1 g Adriamycin and 5 g mAb were administered to each patient. The limiting factor appeared to be a variable dissociation of active Adriamycin from the antibody that unpredictably caused hemopoietic depression. Similar findings were noted among 19 patients treated with mitomycin-C conjugates. Thrombocytopenia at a 60-mg dose of mitomycin-C in this schedule was dose limiting. Serological evidence suggested that the development of an immunoglobulin M antibody specific against the mouse mAb had the specificity and sensitivity to predict clinical reactions. These antibodies were quantitatively less in mitomycin-C-treated patients. Selected patients were retreated. One patient with chronic lymphocytic leukemia was treated on three occasions with regression of peripheral lymph nodes. Two patients with breast carcinoma had definite improvement in ulcerating skin lesions, and two patients with tongue carcinoma had shrinkage of their lesions. No responses were seen with mitomycin-C conjugates but binding was noted to tumors. Drug-induced colitis was seen at higher doses with some binding of these conjugates to normal colon epithelium. This study demonstrated the feasibility of preparing individually specified drug immunoconjugate cocktails for patients with refractory malignancies. Cocktail formulation and antibody delivery to the tumor in vivo was accomplished. There was limited antigenic drift among various biopsies within the same patient over time. The major technical hurdle continues to be the selection of effective drug conjugation methods to optimally bind drugs to mAbs for targeted cancer therapy.
Mol Biother 1991 Sep
PMID:Custom-tailored drug immunoconjugates in cancer therapy. 176 66

A search of compounds capable of inducing specific gene expression in plants without affecting growth and development led to the examination of changes in the pattern of gene expression in corn after treatment with substituted benzenesulfonamide herbicide safeners. Following hydroponic treatment of corn with the safener N-(aminocarbonyl)-2-chlorobenzenesulfonamide (2-CBSU), the specific induction of new translatable mRNA species was observed. Replicate copies of a cDNA library made using RNA from 2-CBSU-treated corn roots were differentially screened with cDNA probes made from either the same mRNA fraction used for library construction or mRNA isolated from roots treated with 2-chlorobenzenesulfonamide (2-CBSA), an inactive analog of the safener. Colonies showing hybridization only with the probe made using mRNA from 2-CBSU-treated roots were further characterized to assess the specificity of the induction and decay of the corresponding induced RNA species. RNA blot analyses showed two clones, designated In2-1 and In2-2, contained plasmids that hybridized to RNAs that were induced from an undetectable background in corn roots within 30 minutes after treatment with 2-CBSU. Leaf and meristem tissues showed similar inductions of the In2-1 and In2-2 RNA species after a delay of several hours. In addition, both RNA species were induced in corn by foliar application of 2-CBSU. In contrast, neither RNA species was induced following stress treatments of plants. These results indicate a substituted benzenesulfonamide safener might be used with the promoters from the In2-1 and In2-2 genes to develop a new inducible gene expression system for plants.
Plant Mol Biol 1991 Oct
PMID:Isolation and characterization of cDNA clones for RNA species induced by substituted benzenesulfonamides in corn. 191 92

The mitochondrial gene, T-urf13, which is unique to the T-cytoplasm of maize, has been expressed in tobacco plants using the Cauliflower Mosaic Virus 35S promoter. Tobacco plants expressing T-urf13 exhibit a variety of responses to methomyl. Leaf discs and petiole sections bleach when exposed to methomyl or HmT-toxin; this effect increases with the age of the tissue. The bleaching effect is not however observed when light is excluded. Plants homozygous for T-urf13 exhibit extreme sensitivity when sprayed with methomyl. The growth of seedling which are either homozygous or heterozygous for T-urf13 is inhibited by methomyl and by kanamycin, whereas seedlings from untransformed tobacco or tobacco which has lost the T-urf13 gene through segregation are sensitive to kanamycin but develop normally when exposed to methomyl. The results demonstrate that T-URF13 need not be specifically targeted to the mitochondrion for it to induce methomyl or HmT-toxin sensitivity in tobacco.
Mol Gen Genet 1991 Oct
PMID:Transfer of methomyl and HmT-toxin sensitivity from T-cytoplasm maize to tobacco. 194 29

The crystal structure of the DNA decamer C-C-A-A-C-G-T-T-G-G has been solved to a resolution of 1.4 A, and is compared with the 1.3 A structure of C-C-A-A-G-A-T-T-G-G and the 1.6 A structure of C-C-A-G-G-C-C-T-G-G. All three decamers crystallize isomorphously in space group C2 with five base-pairs per asymmetric unit, and with decamer double helices stacked atop one another along the c axis in a manner that closely approximates a continuous B helix. This efficient stacking probably accounts for the high resolution of the crystal data. Comparison of the three decamers reveals the following. (1) Minor groove width is more variable than heretofore realized. Regions of A.T base-pairs tend to be narrower than average, although two successive A.T base-pairs alone may not be sufficient to produce narrowing. The minor groove is wider in regions where BII phosphate conformations are opposed diagonally across the groove. (2) Narrow regions of minor groove exhibit a zig-zag spine of hydration, as was first seen in C-G-C-G-A-A-T-T-C-G-C-G, whereas wide regions show two ribbons of water molecules down the walls, connecting base edge N or O with sugar O-4' atoms. Regions of intermediate groove width may accommodate neither pattern of hydration well, and may exhibit a less regular pattern of hydration. (3) Base-pair stacking is virtually identical at equivalent positions in the three decamers. The unconnected step from the top of one decamer helix to the bottom of the next helix is a normal helix step in all respects, except for the absence of connecting phosphate groups. (4) BII phosphate conformation require the unstacking of the two bases linked by the phosphate, but do not necessarily follow as an inevitable consequence of unstacking. They have an influence on minor groove width as noted in point (1) above. (5) Sugar ring pseudorotation P and main-chain torsion angle delta show an excellent correlation as given by the equation: delta = 40 degrees cos (P + 144 degrees) + 120 degrees. Although centered around C-2'-endo, the conformations in these B-DNA helices are distributed broadly from C-3'-exo to O-4'-endo, unlike the tighter clustering around C-3'-endo observed in A-DNA oligomer structures.
J Mol Biol 1991 Jan 05
PMID:Structure of the B-DNA decamer C-C-A-A-C-G-T-T-G-G and comparison with isomorphous decamers C-C-A-A-G-A-T-T-G-G and C-C-A-G-G-C-C-T-G-G. 198 77


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