A method for the quantitative recovery of proteins from polyacrylamide gels.
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Acrylic Resins↗
Biomedical subjects
C A Ryan
Publications and source records attributed to C A Ryan.
RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development.
A 5-kDa polypeptide was isolated from tobacco leaves that induced a rapid alkalinization of the culture medium of tobacco suspension-cultured cells and a concomitant activation of an intracellular mitogen-activated protein kinase. An N-terminal sequence was obtained, and a cDNA coding for the 49-aa polypeptide was isolated from a tobacco cDNA library. The cDNA encoded a preproprotein of 115 amino acids that contained the polypeptide at its C terminus. A search among known expressed sequence tags revealed that genes encoding Rapid ALkalinization Factor (RALF) preproproteins were present in various tissues and organs from 16 species of plants representing 9 families. A tomato homolog of the polypeptide was synthesized and, when supplied to germinating tomato and Arabidopsis seeds, it caused an arrest of root growth and development. Although its specific role in growth has not been established, the polypeptide joins the ranks of the increasing number of polypeptide hormones that are known to regulate plant stress, growth, and development.
Amino Acid Sequence↗
Production of multiple plant hormones from a single polyprotein precursor.
Some animal and yeast hormone genes produce prohormone polypeptides that are proteolytically processed to produce multiple copies of hormones with the same or different functions. In plants, four polypeptides have been identified that can be classed as hormones (intercellular chemical messengers) but none are known to be produced as multiple copies from a single precursor. Here we describe a polyprotein hormone precursor, present in tobacco plants, that gives rise to two polypeptide hormones, as often found in animals and yeast. The tobacco polypeptides activate the synthesis of defensive proteinase-inhibitor proteins in a manner similar to that of systemin, an 18-amino-acid polypeptide found in tomato plants. The two tobacco polypeptides are derived from each end of a 165-amino-acid precursor that bears no homology to tomato prosystemin. The data show that structurally diverse polypeptide hormones in different plant species can serve similar signalling roles, a condition not found in animals or yeast.
Amino Acid Sequence↗
Proteinase inhibitors I and II from potatoes block UVB-induced AP-1 activity by regulating the AP-1 protein compositional patterns in JB6 cells.
Proteinase inhibitor I (Inh I) and proteinase inhibitor II (Inh II) from potato tubers are effective proteinase inhibitors of chymotrypsin and trypsin. Inh I and Inh II were shown to suppress irradiation-induced transformation in mouse embryo fibroblasts suggesting that they possess anticarcinogenic characteristics. We have previously demonstrated that Inh I and Inh II could effectively block UV irradiation-induced activation of transcription activator protein 1 (AP-1) in mouse JB6 epidermal cells, which mechanistically may explain their anticarcinogenic actions. In the present study, we investigated the effects of Inh I and Inh II on the expression and composition pattern of the AP-1 complex following stimulation by UV B (UVB) irradiation in the JB6 model. We found that Inh I and Inh II specifically inhibited UVB-induced AP-1, but not NFkappaB, activity in JB6 cells. Both Inh I and Inh II up-regulated AP-1 constituent proteins, JunD and Fra-2, and suppressed c-Jun and c-Fos expression and composition in bound AP-1 in response to UVB stimulation. This regulation of the AP-1 protein compositional pattern in response to Inh I or Inh II may be critical for the inhibition of UVB-induced AP-1 activity by these agents found in potatoes.
Base Sequence↗
Plant biology: night moves of pregnant moths.
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Animals↗
Radiological case of the month. Perforation of the inferior cava as a cause of neonatal free intra-abdominal air.
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Catheterization, Peripheral↗
Healing response to the Clamshell device for closure of intracardiac defects in humans.
The healing response to intracardiac devices in humans is largely unknown. During regulatory trials using the Clamshell device in over 800 patients, attempts were made to perform histopathological evaluation of all explanted devices. We reviewed all those with complete histopathological examination (n = 12) from Fontan baffles (n = 4), ventricular septal defects (n = 2), and atrial septal defects (ASD; n = 6), explanted at 2.7 months to 3.6 years (median, 1.6 years), at autopsy (n = 1) or surgery (n = 11), performed for residual defects (n = 5), atrial masses (n = 3), or Fontan revision (n = 3). All but one were nearly (n = 3) or completely (n = 8) covered by pseudointima, composed of fibroelastic tissue, predominantly collagen, with focal foreign body reaction in contact with fabric, without acute inflammation or infection. Atrial masses of granulation tissue were present in three cases (ASD), opposite to protruding fractured arms. No associations were identified between coverage and closure status, position, arm fractures, or implant period. In conclusion, the healing response to transcatheter Clamshell implantation in humans is characterized by a relatively rapid development of a nonthrombotic pseudointima composed of fibroelastic tissue with minimal foreign body reaction. Cathet Cardiovasc Intervent 2001;54:101-111.
Adolescent↗
Cytokine induction of a human acute myelogenous leukemia cell line (KG-1) to a CD1a+ dendritic cell phenotype.
Dendritic cells (DC) are highly specialized antigen-presenting cells located in many nonlymphoid tissues, and Langerhans cells (LC), a specialized form of DC, are found in the skin. LC as antigen-presenting cells play a critical role in the induction of allergic contact dermatitis. LC research is difficult because few LCs can be isolated from human skin, so efforts have focused on obtaining DCs from alternative sources. Mononuclear cells from peripheral blood and CD34+ stem cells from human cord blood and marrow can be induced to form phenotypic and functional DCs, but experiments of this type are expensive and the DC yield is low. We report here the induction of the myeloid leukemia cell line (KG-1) to a DC morphology and phenotype by culturing the cells in a defined cytokine cocktail. Morphologically, the KG-1-derived DCs are large irregularly shaped cells with prominent dendritic processes and hair-like cytoplasmic projections. Phenotypically, the KG-1-derived DCs lack lineage-specific markers, and express MHC class II, costimulatory molecules CD80 and CD86, and CD83. Functionally, KG-1-derived DCs are capable of phagocytosing latex microspheres and are able to induce a potent allogeneic T-cell response. Within the KG-1-derived DCs, a subpopulation maintains the DC phenotype and morphology described above but further develops CD1a+ marker expression similar to that of resident skin-derived LCs. These findings illustrate that phenotypic, morphologic and functional DCs can be derived from the KG-1 cell line.
Antigens, CD1↗
Characterization and localization of a wound-inducible type I serine-carboxypeptidase from leaves of tomato plants (Lycopersicon esculentum Mill.).
During the course of characterization of the wound-response related proteins from tomato (Lycopersicon esculentum Mill.) leaves, a serine carboxypeptidase (EC 3.4.16.1) was identified. An increase in peptidase activity in response to wounding, and the isolation of a protein with carboxypeptidase (CP) activity from tomato leaves had been reported previously, but the mRNA coding for the enzyme was not identified. We now report the isolation of a tomato leaf type I serine-CP cDNA whose corresponding mRNA is induced by wounding, systemin and methyl jasmonate. The protein sequence deduced from the cDNA exhibits homology to tomato CP, and barley and rice type I CPs. Southern blot results indicated that the CP gene is probably a member of a small gene family. Tomato CP mRNA was detected within 3 h after wounding, or treatment with systemin or methyl jasmonate. Employing Western blot analysis, CP protein was shown to increase 12 h after the treatments. Using the tomato def1 mutant, we have demonstrated that a functional octadecanoid pathway is necessary for CP transcription in response to wounding. Carboxypeptidase protein was immunolocalized as protein aggregates within the central vacuoles of palisade mesophyll cells as well as in vascular parenchyma where it had previously been found. Double labeling using antibodies specific for CP and inhibitor II indicated that the two proteins are colocalized in the vacuolar aggregates. Tomato CP is a member of the "late wound-inducible genes" whose mRNAs increase 4-12 h following wounding, in contrast to several "early wound-inducible genes", whose mRNAs appear within 30 min. The data support a role for the enzyme in protein turnover that occurs systemically in leaf cells in response to wounding.
Amino Acid Sequence↗
High rate of bacterial vaginosis among women with intrauterine devices in Manado, Indonesia.
Recent research reported that bacterial vaginosis (BV) might enhance the acquisition and transmission of HIV. BV is also associated with an increased risk of pelvic inflammatory disease, a disease also associated with intrauterine device (IUD) insertion. To measure the magnitude of this problem, we conducted a prevalence survey of BV and sexually transmitted diseases (STDs; defined as current infections with Neisseria gonorrhoeae, Chlamydia trachomatis, and/or Trichomonas vaginalis) among all patients attending a family planning clinic in Manado from May to July 1999. BV was diagnosed by Gram stain using Nugent's criteria and vaginal trichomoniasis by wet mount or culture. Cervical infections with C. trachomatis and N. gonorrhoeae were diagnosed by DNA probe. Of 357 patients, 116 (32.5%) had BV, 83 (23.3%) had trichomoniasis, 9 (2.5%) had chlamydia, and 8 (2.2%) had gonorrhea. The prevalence of STD was similar among users of all types of contraception. However, BV was more common among IUD users (47.2%) than among non-IUD users (29.9%). This association persisted after controlling for age, education, ever had douching, and any STD (odds ratio 2.0, 95% CI 1.1-3.8). BV was also associated with STD (41.3% in women with STD vs. 29.4% in women without). This association remained significant after adjusting for age, education, ever had douching, and IUD use (odds ratio 1.7, 95% CI 1.1-2.9). Because we found that BV was associated with IUDs and that other studies reported that both BV and IUDs were associated with pelvic inflammatory disease, a Gram stain evaluation of BV may be considered prior to IUD insertion
Adult↗
Approaches for the development of cell-based in vitro methods for contact sensitization.
Allergic contact dermatitis (ACD) is a cell-mediated immune response to small molecular weight chemicals that contact and penetrate the skin. There are a variety of characteristics that determine whether a chemical can function as a contact sensitizer (or allergen) including the ability to penetrate into the skin, react with protein and be recognized as antigenic by immune cells. The ultimate challenge for developing non-animal test methods for skin sensitization testing will be applying our mechanistic understanding of ACD to the design of predictive in vitro alternative test methods. Specifically, the in vitro approach should be designed so that a chemical's potential to penetrate the skin, react with protein/peptide (biotransformation may be required) and initiate an antigen-specific immune response is incorporated in the test methods developed. In this review, we have focused on cellular-based assays that have been developed or proposed for assessing a chemical's skin sensitization potential in vitro. All of the promising leads to date are based on observations made from in vivo studies conducted in animals and humans, and therefore have a strong mechanistic foundation. However, it remains to be demonstrated whether a single in vitro test, or several in vitro tests in combination, which model the critical steps in sensitization, can replace animal experiments for predicting contact allergic reactions in humans. Regardless, the future looks promising with continued development of our understanding of the chemical and biological aspects of allergic contact dermatitis, and most importantly, with the application of genomics/proteomics to this field on the immediate horizon.
Allergens↗
The suitability of hexyl cinnamic aldehyde as a calibrant for the murine local lymph node assay.
The murine local lymph node assay (LLNA) for the prospective identification of contact allergens assesses skin sensitization potential as a function of proliferative activity induced in lymph nodes draining the site of topical exposure to test chemical. This method has been endorsed recently as a stand alone test for the identification of contact allergens. We have now examined the suitability of hexyl cinnamic aldehyde (HCA), a recommended positive control for skin sensitization testing, as a calibrant for comparing the consistency of LLNA responses with time, and between laboratories, and thus for the routine assessment of assay reliability. Standard LLNAs were performed with CBA strain mice in 3 independent laboratories over a period of 8 years. Dose-response curves were used to derive mathematically the EC3 value (the estimated concentration of chemical necessary to cause a stimulation index (SI) of 3 compared with proliferation induced by concurrent vehicle controls). In each laboratory, 6 separate experiments were conducted using a single concentration of HCA (25%). Very similar stimulation indices were achieved, with mean values of 9.0, 6.5 and 6.6 recorded. A total of 10 dose-response experiments were performed independently in the 3 laboratories and these revealed that there was very little inter-laboratory, or temporal, variation in EC3 values. These data confirm that HCA responses in the LLNA are very stable and demonstrate that HCA provides a suitable calibrant for determining assay sensitivity and performance.
Acrolein↗
Human potency predictions for aldehydes using the local lymph node assay.
The murine local lymph node assay (LLNA) assesses skin sensitization potential as a function of proliferative responses induced in lymph nodes draining the site of topical exposure to test chemical. It has been shown that interpolation of LLNA dose-response data to define the concentration of test chemical required to induce a 3-fold stimulation of proliferation (EC3) offers the prospect of a quantitative index of the relative potency of a contact allergen. Initial studies have demonstrated that there exists a strong (inverse) correlation between EC3 values and contact allergenic potency in humans. Thus, materials with a low EC3 value were more potent contact allergens in humans. However, it is necessary to examine a wide range of allergens to demonstrate that such correlations are generally true. Thus, in the present study, 10 aldehydes of varying degrees of allergenicity in man were evaluated in the LLNA and their EC3 values derived. Formaldehyde was regarded as the strongest allergen in man and also had the lowest EC3 value, 0.35% (equivalent to 0.93% formalin). In contrast, the extremely weak allergen vanillin and the non-sensitizer ethyl vanillin both had EC3 values of >50%. For the remaining 7 aldehydes, there was a close similarity between what is judged to be their rank order of allergenicity in humans and EC3 values derived from analysis of LLNA data. These results support further the utility of EC3 determinations in the LLNA as a measure of the relative potency of a contact allergen.
Aldehydes↗
Contact allergenic potency: correlation of human and local lymph node assay data.
BACKGROUND: Effective toxicologic evaluation of skin sensitization requires that potential contact allergens are identified and that the likely risks of sensitization among exposed populations are assessed. By definition, chemicals that are classified as contact sensitizers have the capacity to cause allergic contact dermatitis (ACD) in humans. However, this hazard is not an all-or-nothing phenomenon; clear dose-response relationships can be discerned and thresholds identified for both the induction of sensitization and the elicitation of ACD. Commonly, these parameters are grouped under the heading of potency, the determination of which is vital for risk assessment. Preclinical testing for sensitization potential is critically important for hazard assessment before human exposure. The murine local lymph node assay (LLNA) is the most recently accepted test method for sensitization hazard assessment. OBJECTIVE: The aim was to compare potency estimations derived from LLNA data with clinical determinations of relative potency based on human data. METHODS: No-effect levels (NOELs) for a range of 21 chemicals were determined from nondiagnostic human repeat patch test studies as reported in the literature. These levels were compared with LLNA EC(3) values, the estimated concentration required to produce a 3-fold increase (positive response) in draining lymph node cell (LNC) proliferative activity. RESULTS: Using available human repeat patch test data, together with expert judgment, the compounds were classified as strong, moderate, weak, extremely weak, or nonsensitizing. Additionally, the potency of each chemical was classified independently based on its LLNA EC(3) value. The results show clearly that LLNA EC(3) values are very comparable with the NOELs calculated from the literature. Moreover, the potency rankings based upon LLNA EC(3) data support their human classification. CONCLUSION: The present investigations show that the LLNA can be used to provide quantitative estimates of relative skin sensitizing potency EC(3) values that correlate closely with NOELs established from human repeat patch testing and from our clinical experience.
Allergens↗
Polypeptide hormones.
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Solanum lycopersicum↗
Wound-inducible proteinase inhibitors in pepper. Differential regulation upon wounding, systemin, and methyl jasmonate.
Seven small (approximately 6,000 D) wound-inducible proteinase inhibitor proteins were isolated from leaves of pepper (Capsicum annuum) plants that are members of the potato inhibitor II family. N-terminal sequences obtained indicated that the pepper leaf proteinase inhibitors (PLPIs) exhibit homology to two GenBank accessions that code for preproteins containing three isoinhibitors domains each that, when post-translationally processed, can account for the mixture of isoinhibitors that are reported herein from pepper leaves. A constitutive level of PLPI proteins was found in pepper leaves, and these levels increased up to 2.6-fold upon wounding of the lower leaves. Exposing intact plants to methyl jasmonate vapors induced the accumulation of PLPIs. Supplying excised young pepper plants with water through the cut stems induced PLPI proteins to levels higher than those found in intact plants, but with high variability. Supplying the excised plants with systemin did not result in an increase of PLPI levels that were statistically higher than levels found in excised plants. Gel-blot analyses of PLPI induction revealed the presence of two mRNA bands, having slightly different mobilities in agarose gels. Only the low M(r) mRNA is present in untreated control plants, and it appears to be responsible for the constitutive levels of PLPI found in leaves. Both mRNA species are wound- and methyl jasmonate-inducible. Only the low- M(r) species is weakly induced by systemin, indicating a differential expression of the two PLPI species.
Acetates↗
Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate.
The systemic accumulation of both hydrogen peroxide (H(2)O(2)) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H(2)O(2)-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H(2)O(2) was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H(2)O(2) acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H(2)O(2) signals for systemic signaling in tomato plants in response to wounding.
Acetates↗
Suramin inhibits initiation of defense signaling by systemin, chitosan, and a beta-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells.
Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and beta-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog (125)I-Tyr-2, Ala-15-systemin to the systemin receptor with an IC(50) of 160 microM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptor-mediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspension-cultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemin-induced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding.
Chitin↗