Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells.

Cancer cells often acquire a constitutively active nuclear factor-kappaB (NF-kappaB) program to promote survival, proliferation and metastatic potential by mechanisms that remain largely unknown. Extending observations from an immunologic setting, we demonstrate that microRNA-146a and microRNA-146b (miR-146a/b) when expressed in the highly metastatic human breast cancer cell line MDA-MB-231 function to negatively regulate NF-kappaB activity. Lentiviral-mediated expression of miR-146a/b significantly downregulated interleukin (IL)-1 receptor-associated kinase and TNF receptor-associated factor 6, two key adaptor/scaffold proteins in the IL-1 and Toll-like receptor signaling pathway, known to positively regulate NF-kappaB activity. Impaired NF-kappaB activity was evident from reduced phosphorylation of the NF-kappaB inhibitor IkappaBalpha, reduced NF-kappaB DNA-binding activity and suppressed expression of the NF-kappaB target genes IL-8, IL-6 and matrix metalloproteinase-9. Functionally, miR-146a/b-expressing MDA-MB-231 cells showed markedly impaired invasion and migration capacity relative to control cells. These findings implicate miR-146a/b as a negative regulator of constitutive NF-kappaB activity in a breast cancer setting and suggest that modulating miR-146a/b levels has therapeutic potential to suppress breast cancer metastases.

Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex.

Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.

Targeting the ets binding site of the HER2/neu promoter with pyrrole-imidazole polyamides.

Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(-1) to 1.4. 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.

Ets regulation of the erbB2 promoter.

Evaluating the chromatinized erbB2 gene in nuclei from breast cancer cells expressing varying levels of ErbB2 transcripts, we identified a nuclease-sensitive site within a 0.22 kb region of maximum enhancer activity centered over a conserved 28 bp polypurine(GGA)-polypyrimidine(TCC) mirror-repeat and an adjacent essential Ets binding site (EBS). Promoter footprinting with nuclear extracts reveals an intense Ets hypersensitivity site at the EBS whose degree of intensity correlates with the level of cellular ErbB2 expression. In vitro mapping assays show that the supercoiled erbB2 promoter forms an internal triplex structure (Hr-DNA) at the mirror-repeat element. Mutations preventing Hr-DNA formation can enhance erbB2 promoter activity in human breast cancer cells, a result consistent with previous demonstration that Ets-erbB2 promoter complexes cannot form when the mirror-repeat is engaged in triplex binding, and new results suggesting that Ets binding induces severe promoter bending that may restrict local triplex formation. In addition to previously described erbB2-regulating breast cancer Ets factors (PEA3, ESX/Elf-3), Elf-1 is now shown to be another endogenously expressed Ets candidate capable of binding to and upregulating the erbB2 promoter. Given current strategies to transcriptionally inhibit ErbB2 overexpression, including development of novel erbB2 promoter-targeted therapeutics, an EBS-targeted approach is presented using chimeric Ets proteins that strongly repress erbB2 promoter activity.

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms.

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.

The synthesis and antibacterial activity of totarol derivatives. Part 1: modifications of ring-C and pro-drugs.

A series of analogues of, and potential pro-drugs derived from, the potent antibacterial diterpene totarol (1) were synthesized in order to elucidate the minimum structural requirements for antibacterial activity and to seek compounds with good bioavailability in vivo. These analogues varied in the structural features of their aromatic rings and the prodrugs were O-glycosylated derivatives. They were tested in vitro against three gram-positive bacteria: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA); and against the gram-negative multi-drug-resistant Klebsiella pneumoniae. None of the analogues was more potent than totarol itself, which is effective against these gram-positive bacteria at MIC values of 7 microM. The results were evaluated in terms of a structure-activity relationship and this showed that a phenolic moiety was essential for potent antibacterial activity. Amongst the pro-drugs, totaryl alpha-D-mannopyranoside (22) proved the most active in vitro (MIC 18 microM). The in vivo antibacterial activities of compounds 1, 22 and totarol beta-lactoside (23) were assessed in a mouse model of infection, but they were found to be ineffective. Compounds 1 and 22 were shown to be cytotoxic towards proliferating human cell cultures, CH 2983, HeLa, and MG 63, but only at concentrations of > 30 microM.

Exon 4-encoded acidic domain in the epithelium-restricted Ets factor, ESX, confers potent transactivating capacity and binds to TATA-binding protein (TBP).

The Ets gene family has a complex evolutionary history with many family members known to regulate genetic programs essential for differentiation and development, and some known for their involvement in human tumorigenesis. To understand the biological properties associated with a recently described epithelium-restricted Ets factor ESX, an 11 kb fragment from the 1q32.2 genomically localized human gene was cloned and analysed. Upstream of the ESX promoter region in this genomic fragment lies the terminal exon of a newly identified gene that encodes a ubiquitin-conjugating enzyme variant, UEV-1. Tissues expressing ESX produce a primary 2.2 kb transcript along with a 4.1 kb secondary transcript arising by alternate poly(A) site selection and uniquely recognized by a genomic probe from the 3' terminal region of the 11 kb clone. Endogenous expression of ESX results in a 42 kDa nuclear protein having fivefold greater affinity for the chromatin-nuclear matrix compartment as compared to other endogenous transcription factors like AP-2 and the homologous Ets factor, ELF-1. Exon mapping of the modular structure inferred from ESX cDNA and construction of GAL4(DBD)-ESX expression constructs were used to identify a transactivating domain encoded by exon 4 having comparable potency to the acidic transactivation domain of the viral transcription factor, VP16. This exon 4-encoded 31 amino acid domain in ESX was shown by mutation and deletion analysis to possess a 13 residue acidic transactivation core which, based on modeling and circular dichroism analysis, is predicted to form an amphipathic alpha-helical secondary structure. Using recombinant GST-ESX (exon 4) fusion proteins in an in vitro pull-down assay, this ESX transactivation domain was shown to bind specifically to one component of the general transcription machinery, TATA-binding protein (TBP). Transient transfection experiments confirmed the ability of this TBP-binding transactivation domain in ESX to squelch heterologous promoters independent of any promoter binding as efficiently as the transactivation domain from VP16.

Increased activator protein-1 DNA binding and c-Jun NH2-terminal kinase activity in human breast tumors with acquired tamoxifen resistance.

Human breast tumors that are initially responsive to tamoxifen (TAM) eventually relapse during treatment. Estrogen receptor (ER) expression and function are often preserved in these tumors, and clinical evidence suggests that this relapse may be related to TAM's known agonistic properties. ER can interact with the activator protein-1 (AP-1) transcription factor complex through protein-protein interactions that are independent of ER DNA binding and, in certain ER-positive cells, this may allow TAM to exert an agonist response on AP-1-regulated genes. We, therefore, assessed both AP-1 DNA binding and the known AP-1 activating enzyme, c-Jun NH2-terminal kinase (JNK), in a panel of 30 ER-positive primary human breast tumors with acquired TAM resistance, as compared to a matched panel of 27 untreated control ER-positive breast tumors and a separate control set of 14 primary tumors, which included 7 ER-positive tumors that were growth-arrested by 3 months of preoperative TAM. AP-1 DNA binding activity was measured from cryopreserved tumor extracts using a labeled oligonucleotide probe containing a consensus AP-1 response element by electrophoretic mobility shift assay. JNK was first extracted from the tumor lysates by incubation over a Sepharose-bound c-Jun(1-89) fusion protein, and its activity was then measured by chemiluminescent Western blot by detection of the phosphorylated product using a phospho-Jun(Ser-63)-specific primary antibody. The set of control ER-positive breast tumors growth arrested by TAM showed no significant difference from untreated control tumors in their AP-1 DNA binding and JNK activities. In contrast, there was a significant (P < 0.001) increase in mean AP-1 DNA binding activity for the panel of ER-positive TAM-resistant (TAM-R) tumors as compared to its matched control panel of untreated tumors. Mean JNK activity in the TAM-R tumors was also significantly higher than that found in the untreated tumors (P = 0.038). Overall, there was no significant correlation between JNK activity and AP-1 DNA binding; however, regression analysis showed that, for any given level of JNK activity, the TAM-R tumors possessed a 3.5-fold increase in AP-1 DNA binding activity as compared to the untreated tumors. These findings indicate that, when compared to untreated ER-positive primary breast tumors, TAM-R tumors demonstrate significantly increased levels of AP-1 DNA binding and JNK activity, consistent with experimental models suggesting that TAM-stimulated ER-positive tumor growth may be mediated by enhanced AP-1 transcriptional activity. These observations support the need for further evaluation of these markers in breast tumors as predictors of TAM resistance.

The epithelium-specific ets transcription factor ESX is associated with mammary gland development and involution.

To study mammary gland expression of the epithelium-restricted Ets factor, ESX, mouse cDNA and genomic sequences were cloned and a approximately 350 bp proximal promoter region with >80% mouse-human homology was identified that mediates ESX induction by serum, heregulin (HRG), or epidermal growth factor (EGF). ESX mRNA expression progressively increases during embryonic mouse development from day 7 and is detectable in virgin mammary glands; it shows little if any change during pregnancy, then declines to barely detectable levels after 3 days of lactation. Similarly, cultured HC11 cells from midpregnant mouse mammary epithelium show an increase in ESX expression upon reaching lactogenic competency (in the presence of EGF or HRG), with a decline to barely detectable levels upon exposure to lactogenic hormones that induce milk protein (beta-casein) expression. In contrast, involuting mouse and rat mammary glands show maximal ESX expression. High ESX levels are also seen in the involuting ventral prostate gland of rats. These findings, including the persistence of up-regulated ESX in fully regressed mammary glands, suggest that ESX expression can be induced by soluble growth factors and is maximally up-regulated in those partially committed epithelial cells destined to survive both the apoptotic and remodeling phases of glandular involution.

Polyamine inhibition of estrogen receptor (ER) DNA-binding and ligand-binding functions.

Polyamines are known to inhibit sequence specific DNA-binding activity of several zinc-finger transcription factors, including estrogen receptor (ER) binding to its cognate estrogen response element (ERE). The mechanism accounting for this disruption of protein-DNA interaction is unknown, although polyamine induction of DNA conformational changes has been suggested. To determine if polyamines can directly impair ER action, we compared the effects of putrescine (Putr), spermidine (Spd), and spermine (Spm) on ER DNA-binding (ER-ERE complex formation), ER ligand-binding (estradiol), ER structure (circular dichroism and sucrose gradient sedimentation), and the capacity of ER to transactivate an ERE-tk-CAT reporter in transient transfection assays. Polyamine concentrations causing 50% inhibition of ER-ERE formation (IC50 values) were found to be 1 mM for Putr, 4 mM for Spd, and 3 mM for Spm. This loss of ER DNA-binding was associated with a direct and irreversible effect on the ER DNA-binding domain (ER-DBD). Additionally, polyamines were observed to inhibit ER ligand-binding with IC50 values of 10 mM for Putr, 2 mM for Spd, and < 0.1 mM for Spm; and this correlated with a measureable change in higher-order ER structure (5S to 3.5S sedimentation) and inhibition of intracellular ER transactivation. These findings suggest that in ER-positive human breast tumors with increased polyamine (especially Spm) content, ER structure and function may be directly altered by tight-ion polyamine complexing that results in loss of ER-mediated gene regulation.

Oxidant stress impaired DNA-binding of estrogen receptor from human breast cancer.

Full-length (67 kDa) immunoreactive estrogen receptor (ER) extracted from a third of untreated ER-positive primary breast tumors appears unable to bind to its cognate estrogen response element (ERE). We have observed partial reversibility of this ER DNA-binding defect upon treatment of these tumor extracts with excess thiol reducing agent (DTT), suggesting that ER DNA-binding is subject to redox modulation as is reported for other zinc-finger proteins and transcriptional activators. Treatment of recombinant ER DNA-binding domain (ER-DBD) or ER-enriched extracts from CHO(ER) and MCF-7 cells with thiol-reacting oxidants (diamide, iodosobenzoate, H2O2) or alkylator (iodoacetamide) produces a dose-dependent loss in ER DNA-binding capacity. Thiol-specific oxidative loss in ER DNA-binding is fully reversible by DTT reduction, unlike the defect caused by thiol-specific alkylation. Circular dichroism spectrometry shows that both forms of treatment substantially modify ER secondary structure, inducing loss of alpha-helical content within the ER-DBD that is reversible after thiol oxidation but not after thiol alkylation. Oxidant (H2O2, menadione) exposure of cultured CHO(ER) or MCF-7 cells impairs the ability of endogenous ER to bind DNA and transactivate an ER-responsive reporter gene (ERE-tk-CAT), demonstrating that extracellular redox stress can modulate intracellular ER function. Since these thiol-specific oxidant and alkylator treatments have no significant effect on either recombinant ER ligand-binding or intracellular immunoreactive ER content, our findings suggest that DNA-binding and transactivation are the most sensitive intracellular ER functions impaired by oxidant stress in some ER-positive human breast tumors.

HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer.

HER2/Neu is overexpressed in 25-30% of all human breast cancers as a result of both gene amplification and enhanced transcription. Transcriptional upregulation of HER2/neu leads to a 6-8-fold increased abundance of its mRNA per gene copy and likely results from the elevated activity of transcription factors acting on the HER2/neu promoter. Here we report that transcripts of PEA3, an ETS transcription factor implicated in oncogenesis, were increased in 93% of HER2/Neu-overexpressing human breast tumor samples. Analyses to uncover the molecular basis for elevated PEA3 transcripts in HER2/Neu-positive breast tumors revealed that the HER2/Neu receptor tyrosine kinase initiated an intracellular signaling cascade resulting in increased PEA3 transcriptional activity; transcriptionally-activated PEA3 stimulated HER2/neu and PEA3 gene transcription by binding to sites in the promoters of these genes. PEA3 also activates transcription of genes encoding matrix-degrading proteinases, enzymes required for tumor cell migration and invasion. These findings implicate PEA3 in the initiation and progression of HER2/Neu positive breast cancer, and suggest that PEA3 and signaling proteins affecting its regulation are appropriate therapeutic targets.

Comparison of estrogen receptor DNA binding in untreated and acquired antiestrogen-resistant human breast tumors.

Preliminary studies have suggested that measuring the ability of immunoreactive 67-kDa estrogen receptor (ER) to bind DNA and form in vitro complexes with its cognate estrogen response element (ERE) might serve to identify breast tumors most likely to respond to antiestrogens like tamoxifen. Data from two different surveys of untreated primary breast tumors confirmed that only 67% (74 of 111) of ER-positive tumors express a receptor capable of forming ER-ERE complexes by gel-shift assay, with tumors of lower ER content having significantly reduced ER DNA-binding frequency (56%) relative to those of higher ER content (82%; P = 0.007). In contrast to these untreated tumors, a panel of 41 receptor-positive breast tumors excised after acquiring clinical resistance to tamoxifen during either primary (n = 26) or adjuvant therapy (n = 15) showed a significantly greater ER DNA-binding frequency, with nearly 90% capable of forming ER-ERE complexes (P < 0.02). To assess experimentally whether ER DNA-binding function is altered during the development of antiestrogen resistance, nude mouse MCF-7 tumor xenografts were analyzed before and after the acquisition of in vivo resistance to either tamoxifen or a pure steroidal antiestrogen, ICI 182,780. Tamoxifen-resistant MCF-7 tumors retained full expression of 67-kDa DNA-binding ER, and despite a markedly reduced ER content in the ICI 182,780-treated tumors, the expressed ER in these antiestrogen-resistant tumors exhibited full ability to form ER-ERE complexes. These findings indicate that breast tumors with acquired antiestrogen resistance continue to express ER of normal size and DNA-binding ability and suggest that the failure of antiestrogens to arrest tumor growth during emergence of clinical resistance results from an altered gene-regulatory mechanism(s) other than ER-ERE complex formation.

ESX: a structurally unique Ets overexpressed early during human breast tumorigenesis.

The >30 known members of the Ets multigene family of transcriptional regulators are increasingly being recognized for their involvement in early embryonic development and late tissue maturation, directing stage-specific and tissue-restricted programs of target gene expression. Identifiable primarily by their 85 amino acid ETS DNA-binding domain and dispersed across all metazoan lineages into distinct subfamilies, Ets genes also produce malignancies in humans and other vertebrates when overexpressed or rearranged into chimeras retaining the ETS domain, suggesting that their oncogenic potential is determined by the program of target genes they regulate. Searching for Ets factors that regulate expression of the HER2/neu (c-erbB2) oncogene in human breast cancer, we identified a new epithelium-restricted Ets encoding an ETS domain homologous to the Drosophila E74/human Elf-1 subfamily, an amino-terminal region (A-region or Pointed domain) homologous to the distantly related Ets-1 subfamily, and a serine-rich box homologous to the transactivating domain of the lymphocyte-restricted High Mobility Group (HMG) protein, SOX4. Recombinant protein encoded by ESX (for epithelial-restricted with serine box) exhibits Ets-like DNA binding specificity in electrophoretic mobility shift assays and, in transient transfection assays, transactivates Ets-responsive promoter elements including that found in the HER2/neu oncogene. ESX is located at chromosome 1q32 in a region known to be amplified in 50% of early breast cancers, is heregulin-inducible and overexpressed in HER2/neu activated breast cancer cells. Tissue hybridization suggests that ESX becomes overexpressed at an early stage of human breast cancer development known as ductal carcinoma in situ (DCIS).

Check bite impressions using irreversible alginate/reversible hydrocolloid combination.

A restoration that the patient reports as "high" is a common problem for the restorative dentist. Check bite, dual bite, or closed mouth impressions address this problem effectively by eliminating the opposing arch impression and articulating the opposing cast accurately. In this article a procedure that combines irreversible hydrocolloid and reversible hydrocolloid results in a rapid, clean, and effective solution.

Further evidence for the mitogenic action of urinary trypsin inhibitor.

We have previously shown that urinary trypsin inhibitor (UTI), also known and bikunin, was mitogenic for human fibroblasts at low concentrations, and growth-inhibitory at higher concentrations, and have identified high- and low-affinity cellular binding sites for this protein. We have now investigated fibroblast proteins which interact with bikunin. Bikunin binds to proteins of about 50K and 250K. The simplest interpretation, is that the 50K protein may be a proteinase which is also the low-affinity bikunin binding site, involved in growth inhibition, and that the larger protein may be responsible for the mitogenic response to bikunin. Inhibitors of intracellular calcium mobilisation also inhibit the mitogenic response to bikunin, and by the measurement of the efflux of pre-loaded 45Ca2+, bikunin at mitogenic concentrations can be shown to stimulate calcium mobilization.

Biphasic modulation of cell growth by recombinant human galectin-1.

Human soluble galactose-binding lectin (galectin-1) has been expressed as an Escherichia coli fusion protein, following the amplification by polymerase chain reaction of cDNA prepared from a human osteosarcoma cell line. The fusion protein is a functional beta-galactoside-binding lectin, as is the recombinant galectin when purified from the cleaved fusion protein. The recombinant galectin has a biphasic effect on cell proliferation. Unlike the fusion protein, it functions as a human cell growth inhibitor, confirming earlier findings with natural human galectin-1, though it is less effective than the natural galectin. This reaction is not significantly inhibited by lactose, and is thus largely independent of the beta-galactoside-binding site. At lower concentrations, recombinant galectin-1 is mitogenic, this activity being susceptible to inhibition by lactose, and thus attributable to the beta-galactoside-binding ability of the protein. Some tumour cells are susceptible to the growth-inhibitory effect, and the galectin-1 gene is expressed in both normal and tumour cells.

Evidence of post-transcriptional regulation of U6 small nuclear RNA.

Mechanisms regulating the intracellular level of endogenous U6 small nuclear RNA were studied by transient transfection of ectopic U6 gene constructs into immortalized normal and malignant human cell lines. Transfection and expression of a modified U6 gene containing native promoter, capping, and termination sequences but lacking all highly conserved internal spliceosome sequences produced dose-dependent effects on endogenous U6 gene expression. At low transfection doses, no significant changes in endogenous U6 RNA levels or half-life were noted. However, as the dose of the transfected gene and its expression increased, native U6 RNA levels dramatically decreased in association with an apparent decrease in U6 RNA half-life. Down-regulation of native U6 RNA levels was transient, with recovery noted within 48-96 h in conjunction with declining expression of the ectopic gene. These modulatory effects appeared specific to endogenous U6 transcripts, because no changes were noted in 7sk, U1, U3, or 5S RNA levels or half-lives. Transfection with an unmodified U6 gene did not alter total U6 transcript levels but did produce a similar dose-dependent decrease in U6 RNA half-life. These studies suggest a hitherto unrecognized U6-specific intracellular regulatory mechanism, through which over-accumulation of U6 small nuclear RNA is prevented.

Isolation, characterisation and cell growth-regulatory properties of kumara (sweet potato) trypsin inhibitors.

Locally grown kumara (sweet potato; Ipomea batatis) was used as the starting point for the purification of a proteinase inhibitor. The purified inhibitor was highly specific for trypsin, and much less effective as an inhibitor of chymotrypsin. Two 22kDa variants were present, closely homologous to each other and to sporamin A, but with a single amino acid substitution (proline in place of serine, the second residue in mature sporamin A). One variant had the same N-terminus as sporamin A, whereas the other had a tripeptide N-terminal extension, which may represent an intermediate in the proteolytic processing of the precursor protein. A larger variant was apparently a disulphide-linked dimer of the monomeric inhibitor. A rabbit polyclonal antiserum prepared against the trypsin inhibitor reacted with all of these variants, but did not cross-react with commercially-available soybean proteinase inhibitors. The purified inhibitor did resemble other proteinase inhibitors in having a biphasic effect upon the proliferation of human fibroblasts, with a mitogenic action at low concentrations, and an inhibitory effect at higher concentrations.