Structural studies of reagents for peptide bond formation: Crystal and molecular structures of HBTU and HATU

Summary X-ray structure determinations of HBTU and HATU, well-known reagents for peptide bond formation, show that the solid-state structures of these compounds differ markedly from those commonly presented in the literature. The solid-state structures are isomers of the N,N,N',N'-tetramethyluronium salt formulation commonly written for HBTU and HATU. HBTU and HATU, obtained either directly from synthesis or from CH₃CN solution, crystallize as the guanidinium N-oxide isomers. Both compounds crystallize in nearly identical conformations, despite marked differences in crystal packing.     

Protein expression of a self-compatible allele from Lycopersicon peruvianum: introgression and behavior in a self-incompatible background

Lycopersicon peruvianum (wild tomato) is a gametophytic self-incompatible (SI) species. One natural population has been shown to harbor a self-compatible (SC) allele. A stylar protein associated with the self-compatibility allele has been elucidated using SDS-PAGE. The temporal and spatial expression of this protein is presented and compared with protein expression of two SI alleles. Hybrids containing the SC and SI alleles were used in a backcrossing program to introgress the SC allele into SI backgrounds in six independent lines. Controlled pollinations and SDS-PAGE were used to identify and select classes of progeny. After four backcross generations (approximately 97% recovery of the SI backgrounds) the SC allele still confers self-fertility in lines that contain this allele, providing evidence that the mutation to SC occurred at the S-locus and that the associated protein is likely responsible.     

Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min⁻¹ mg⁻¹. A key to this purification protocol was the introduction of a size-exclusion chromatography step, a size-based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl-Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.     

Cloning and expression of soluble epoxide hydrolase from potato

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.     

The forever young gene encodes an oxidoreductase required for proper development of the Arabidopsis vegetative shoot apex

In plant development, leaf primordia are formed on the flanks of the shoot apical meristem in a highly predictable pattern. The cells that give rise to a primordium are sequestered from the apical meristem. Maintenance of the meristem requires that these cells be replaced by the addition of new cells. Despite the central role of these activities in development, the mechanism controlling and coordinating them is poorly understood. These processes have been characterized in the Arabidopsis mutant forever young (fey). The fey mutation results in a disruption of leaf positioning and meristem maintenance. The predicted FEY protein shares significant homology to a nodulin and limited homology to various reductases. It is proposed that FEY plays a role in communication in the shoot apex through the modification of a factor regulating meristem development.     

A (1→3,1→4)-β-glucan-specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1→3,1→4)-β-glucans

Monoclonal antibodies were raised against a (1→3,1→4)-β-glucan-bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1→3,1→4)-β-glucan, displaying no binding activity against a (1→3)-β-glucan-BSA conjugate and minimal binding against a cellopentaose-BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1→3,1→4)-β-glucan, purified by size exclusion chromatography and characterized by ¹H-NMR and anion exchange chromatography. These (1→3,1→4)-β-oligoglucosides, together with (1→3)-β- and (1→4)-β-oligoglucosides were used to characterize the binding site of the monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody showed maximal binding to a heptasaccharide with the structure Glc(1→3) Glc(1→4) Glc(1→4) Glc(1→3) Glc(1→4) Glc(1→4) Glc and was determined to have an affinity constant of 3.8 × 10⁴ M⁻¹ for this oligoglucoside. The monoclonal antibody (BG1) has been used to develop a sensitive sandwich ELISA for the specific quantitation of (1→3,1→4)-β-glucans. The assay operates in the range 1–10 ng ml⁻¹ and shows no significant cross-reaction with tamarind xyloglucan, wheat endosperm arabinoxylan or carboxymethyl-pachyman ((1→3)-β-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region. A previously described specific anti-(1→3)-β-glucan antibody (Meikle et al., 1991) bound to discrete patches on the aleurone walls, believed to be plasmodesmata.     

Human familial and sporadic breast cancer: analysis of the coding regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand conformation polymorphism assay

17-Hydroxysteroid dehydrogenase (17HSD) is one of the key enzymes in estrogen metabolism, catalyzing the reversible reaction between estradiol and the less active estrogen, estrone. The gene encoding this enzyme, EDH17B2, has been mapped to chromosome 17, region q12–q21, in the vicinity of BRCA1, an as yet unidentified gene that appears to be involved in familial breast cancer and in familial ovarian cancer. The possibility that EDH17B2 gene is the same as BRCA1 was tested by screening for mutations in the coding regions of EDH17B2, using a polymerase chain reaction/single-strand conformation polymorphism method. An AG transition creating a new BstUI site at exon 6 was the only frequent sequence alteration found in the coding region of the gene. This mutation also led to an amino acid substitution of serine to glycine at position 312 (312^S312^G) in the 17HSD protein. Since the nucleotide change was detected both in specimens from patients with familial or sporadic cancer and in control samples, and at similar rates, this mutation appears to be of a polymorphic nature. In addition, a rare polymorphism located at intron 5 was detected. This CT substitution creates a BbvI site and is not thought to have any effect on 17HSD activity. The results indicate that there are no major alterations in the coding areas of EDH17B2 and thus studies testing the hypothesis that EDH17B2 may be the same as BRCA1 should be extended to the promoter and regulatory elements of EDH17B2.     

Effect of interferon γ and tumour necrosis factor α on sensitivity to cisplatin in ovarian carcinoma cell lines

This study aimed to investigate whether the biological response modifiers (BRM) interferon (IFN) and tumour necrosis factor (TNF) could enhance the cytotoxic action of cisplatin on ovarian tumour cells in vitro. The sensitivity of four cell lines (OAW42, GG, JAM and PE01) to drugs and drug combinations was tested by a radiolabelled-thymidine incorporation assay. Cell lines demonstrated a range of sensitivity to cisplatin and the innate cytotoxic effect of each of the BRM. When IFN was used in combination with cisplatin, a significant enhancement of cisplatin toxicity occurred in three of four cell lines. TNF demonstrated such an effect in two cell lines but diminished the toxicity of cisplatin in one cell line. A purely additive effect of the agents may explain the enhanced toxicity of cisplatin in some of these cases. However, in one cell line at least (PEO1), both TNF and IFN demonstrated a clearly synergistic effect with cisplatin. These BRM used in conjunction with cisplatin may provide better antitumour regimen than cisplatin alone in some patients with ovarian cancer, but the response is likely to be heterogeneous between patients.     

Transfection of the mouse ICAM-1 gene into murine neuroblastoma enhances susceptibility to lysis,reduces in vivo tumorigenicity and decreases ICAM-2-dependent killing

We determined the expression of intercellular adhesion molecules (ICAM) on neuro-2a cells in order to evaluate whether they were involved in cytolysis of murine neuroblastoma. Fluorescence-activated cell sorting analysis revealed that the control neomycin-resistance-genetransduced line (neuro-2a/LN) had poor expression of ICAM-1 (mean channel fluorescence, MCF=3.7). An ICAM-1-positive transfectant of neuro-2a (neuro-2a/ICAM-1⁺) (CMF=64.3) was generated to evaluate directly the role of this adhesion molecule in cytolysis. Neuro-2a/ICAM-1⁺ was more sensitive to LAK killing (69.7% at an effector-to-target ratio of 1001) compared to neuro-2a/LN (48.6%) (P<0.001). Blocking of neuro-2a/LN and neuro-2a/ICAM-1⁺ lysis with anti-ICAM-1 monoclonal antibodies (mAbs) did not account for all the LFA-1-dependent killing. These data indicate that even in neuro-2a/ICAM-1⁺ cells, other LFA-1 ligands participated in the effector-target interaction. Therefore, we examined these cell lines for ICAM-2 expression. Both neuro-2a/LN and neuro-2a/ICAM-1⁺ lines expressed ICAM-2 (MCF=16.4 and 16.5). ICAM-2 accounted for the majority of the LFA-1-dependent killing in the ICAM-1-negative target, neuro-2a/LN, while ICAM-1 played a primary role in the cytolysis of the ICAM-1⁺ transfectant. Inhibition of lysis in the presence of anti-ICAM-1 and ICAM-2 mAbs was comparable to that seen with the addition of anti-LFA-1 mAb, indicating that other LFA-1 ligands were not involved in this system. ICAM-1 expression was associated with decreased in vivo tumorigenicity; mice inoculated with neuro-2a/ICAM-1⁺ cells had a significantly longer survival compared to those receiving neuro-2a/LN cells (median survival time 35.5 versus 24.5 days) (P<0.001). It is important to note that ICAM-1 transfection of murine neuroblastoma did not alter its metastatic potential. We conclude that transfection of mouse neuroblastome with ICAM-1 increases its sensitivity to in vitro lysis and reduces its in vivo tumorgenicity. In ICAM-1-negative murine neuroblastoma cells, ICAM-2 plays a primary role in cell-mediated lysis.This work was supported in part by the Children's Cancer Research Fund, the Minnesota Medical Foundation, the Viking Children's Fund and NIH grants PO1-CA-21737, NO1-AI-85002. E. K. is a recipient of the Irvine McQuarrie Research Scholar Award and B. R. B. a recipient of the Edward Mallinkrodt Foundation Scholar Award