A single-chain antibody or single-chain Fv (sFv) incorporates the complete antibody binding site in a single polypeptide chain of minimal size, with an approximate molecular weight of 26,000. In antibodies, the antigen combining site is part of the Fv region, which is composed of the VH and VL variable domains on separate heavy and light chains. Efforts over nearly two decades have indicated that Fv fragments can only rarely be prepared from IgG and IgA antibodies by proteolytic dissection. Beginning in 1988, single-chain analogues of Fv fragments and their fusion proteins have been reliably generated by antibody engineering methods. The first step involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis. The single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly4-Ser)3. The linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH. In principle, the sFv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site, as demonstrated in our model studies with the 26-10 anti-digoxin sFv. Furthermore, the sFv remains stable at low concentrations that promote VH and VL dissociation from the Fv heterodimer, resulting in loss of Fv binding. Intravenously administered sFv proteins exhibit accelerated biodistribution and exceptionally fast clearance compared to IgG or Fab. These pharmacokinetic properties allow rapid imaging by sFv, which therefore may be labeled with a short-lived isotope such as Tc-99m. Expression of a single gene product from fused sFv and effector genes facilitates immunotargeting of the effector protein, as shown for single-chain Fv toxin fusion proteins.

Partial digestion of polygalacturonic acid with polygalacturonase isolated from Rhizopus stolonifer produces a mixture of a-1,4-D-galacturonide oligomers which act to elicit casbene synthetase activity in castor bean (Ricinus communis L). These oligomers were separated by anion exchange chromatography on DEAE Sephadex A-25 into discrete sizes and their degrees of polymerization were analyzed by fast atom bombardment mass spectrometry. A minimum degree of polymerization of nine units appears to be required for elicitor activity, trideca-a-1,4-Dgalacturonide was the most active of the oligomers tested. Methylesterification of the carboxylate groups greatly diminishes the elicitor activity of the oligomers, a finding which suggests a requirement for the polyanionic character of the oligomers for full activity. The fact that a number of other polyanionic polymers tested as casbene synthetase elicitors did not show significant activity indicates that structural features other than the polyanionic character are also necessary for activity.Castor bean (Ricinus communis L.) seedlings respond to challenges from a variety of fungi by producing an antifungal diterpene hydrocarbon, casbene (8). Casbene synthetase activity in castor bean seedlings increases dramaticlly in response to a heatlabile elicitor which was purified to homogeneity from culture filtrates of the fungus, Rhizopus stolonifer. The elicitor activity was found to be associated with the enzyme endopolygalacturonase, an endo a-1,4-galacturonide hydrolase (6), and was subsequently shown to be absolutely dependent on the catalytic activity of this enzyme (2). Several lines of evidence support the idea that heat-stable pectic fragments released from isolated castor bean cell walls through the action of the enzyme serve as obligate intermediates in the elicitation process (2).These results suggested that products released by degradation of the plant cell wall might act as signals for the elicitation of stress metabolites under certain circumstances. A majority of the work on elicitors has focused on molecules of fungal origin as the primary signals in eliciting the plants's response (9). However, recent work in several laboratories has called attention to molecules originating in the plant as elicitors. Hargreaves et al. (4,5) have described the elicitation of phaseollin in hypocotyls of Phaseolus vulgaris in response to 'constitutive metabolites' released from damaged bean cells. The chemical nature of these constitutive elicitors has not been elucidated. Hahn et al. (3) found that partial acid hydrolysis of cell walls of soybeans and other plants released 'endogenous elicitors' that acted to stimulate

Interactions between estrogen and growth factor signaling pathways at the level of gene expression play important roles in the function of reproductive tissues. For example, estrogen regulates transforming growth factor beta (TGF␤) in the uterus during the proliferative phase of the mammalian reproductive cycle. Bone morphogenetic protein 7 (BMP-7), a member of the TGF␤ superfamily, is also involved in the development and function of reproductive tissues. However, relatively few studies have addressed the expression of BMP-7 in reproductive tissues, and the role of BMP-7 remains unclear. As part of an ongoing effort to understand how estrogen represses gene expression and to study its interactions with other signaling pathways, chick BMP-7 (cBMP-7) was cloned. cBMP-7 mRNA levels are repressed threefold within 8 h following estrogen treatment in the chick oviduct, an extremely estrogen-responsive reproductive tissue. This regulation occurs at the transcriptional level. Estrogen has a protective role in many tissues, and withdrawal from estrogen often leads to tissue regression; however, the mechanisms mediating regression of the oviduct remain unknown. Terminal transferase-mediated end-labeling and DNA laddering assays demonstrated that regression of the oviduct during estrogen withdrawal involves apoptosis, which is a novel observation. cBMP-7 mRNA levels during estrogen withdrawal increase concurrently with the apoptotic index of the oviduct. Furthermore, addition of purified BMP-7 induces apoptosis in primary oviduct cells. This report demonstrates that the function of BMP-7 in the oviduct involves the induction of apoptosis and that estrogen plays an important role in opposing this function.

Spermatogenic cells have been previously shown to be a major site of testicular proenkephalin gene expression. Using RNA gel-blot analysis of purified mouse and hamster germ cells and of testes from prepuberal and germ cell-deficient mutant mice, we now have demonstrated that, in addition to its previously described expression by somatic (Leydig) cells, the gene for a second opioid peptide precursor, pro-opiomelanocortin (POMC), is also expressed by spermatogenic cells. Of particular significance is the finding that the RNAs for proenkephalin and POMC are differentially regulated during spermatogenesis. Two forms of POMC RNA were detected in mouse testis, a larger component 675-to 750-nucleotides (nt) in size common to somatic and spermatogenic cells and a smaller 625-nt RNA found only in pachytene spermatocytes. Two distinct, cell-specific proenkephalin RNAs were also shown to be present in mouse testis: a 1700-nt transcript previously shown to be expressed by spermatogenic cells and a 1450-nt form associated with somatic cells. These data suggest that (i) proenkephalin-and PONMC-derived peptides are produced by both somatic cells and germ cells in the testis and (ii) in germ cells these two families of opioid peptides may function at different stages of spermatogenesis.Paracrine mechanisms play an important role in the maintenance and regulation of spermatogenesis within the testis (1-3). Several factors appear to mediate interactions between different testicular cell types, including P Mod-S, a protein produced by peritubular cells that modifies Sertoli cell secretion (4); transport proteins and mitogenic factors produced by Sertoli cells that seem important for germ cell proliferation (3-5); and testosterone produced by Leydig cells and required for normal spermatogenesis (2). The recent demonstration that each of the three opioid peptide precursors, pro-opiomelanocortin (POMC), proenkephalin, and prodynorphin, are expressed in the testis (6-9) suggests that their peptide products also locally regulate testicular function. We have previously shown that spermatogenic cells are a major site of proenkephalin RNA expression in the mouse testis (10), results suggesting that proenkephalin-derived peptides function as germ cell-associated paracrine factQrs. The gene for the opioid peptide precursor POMC has already been shown to be expressed by Leydig cells (6, 7). In this report the POMC gene is demonstrated to be expressed also by testicular germ cells and in a distinct manner during spermatogenesis from that described for the proenkephalin gene. Multiple proenkephalin and POMC transcripts are further shown to be produced in the rodent testis in a cell-type-specific manner. MATERIALS AND METHODSGuanidine thiocyanate and N-lauroylsarcosine were from Fluka Chemical (Happauge, NY), 32P-labeled deoxynucleotides (3000 Ci/mmol; 1 Ci = 37 GBq) were purchased from New England Nuclear, cesium chloride was from Bethesda Research Laboratories, collagenase was obtained from Worthington, and trypsin (pancreatic) and D...

Rhizopus stolonifer endopolyglacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and funpl cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks.