ABSTRACT
Between 1992 and 2017, the Antarctic Ice Sheet (AIS) lost ice equivalent to 7.6 ± 3.9 mm of sea level rise. AIS mass loss is mitigated by ice shelves that provide a buttress by regulating ice flow from tributary glaciers. However, ice-shelf stability is threatened by meltwater ponding, which may initiate, or reactivate preexisting, fractures, currently poorly understood processes. Here, through ground penetrating radar (GPR) analysis over a buried lake in the grounding zone of an East Antarctic ice shelf, we present the first field observations of a lake drainage event in Antarctica via vertical fractures. Concurrent with the lake drainage event, we observe a decrease in surface elevation and an increase in Sentinel-1 backscatter. Finally, we suggest that fractures that are initiated or reactivated by lake drainage events in a grounding zone will propagate with ice flow onto the ice shelf itself, where they may have implications for its stability.
ABSTRACT
BACKGROUND/PURPOSE: Photoaging of the skin is a result of chronic exposure to environmental ultraviolet radiation (UV). The milieu provided by the extracellular matrix, which significantly influences the behaviour of resident fibroblasts, depends critically on the supermolecular collagen structure. We ask whether direct photochemical treatment of type I collagen with solar wavelengths capable of reaching the dermis can modify the substrate's susceptibility to collagenase in a model in vitro system. METHODS: Acid- extracted Skh-1 hairless mouse collagen samples were irradiated with 0-140 J/cm2 of radiation from bank of filtered FS lamp (UVB/UVA = 0.33, fluence rate = 0.81 mW/cm2). Subsequent to UV irradiation, collagen samples were coupled with fluorescein isothiocyanate (FITC) and assayed for susceptibility to bacterial collagenase by monitoring the appearance of supernatant FITC fluorescence (a measure of lysed collagen) over time of incubation. As a 'reference', unirradiated commercial FITC-labelled citrate-soluble collagen (Elastin Products, Owensville, MO 65066, USA) was similarly analysed. RESULTS: Unirradiated mouse collagen had a lower rate of cleavage than did the calfskin sample. Irradiation of unlabelled mouse collagen for 0-48 h (0-140 J/cm2 total UV) rendered the sample more soluble, with concomitant chain degradation, cross-linking and loss of intrinsic collagen fluorescence. At irradiation time's >/= 4 h (>/=11.7 J/cm2), the irradiated collagen was significantly more susceptible to bacterial collagenase digestion. DISCUSSION: It appears that the rate of cleavage depends on the superstructure of the collagen, since the kinetics of collagen cleavage differ for two collagen samples having essentially the same primary structure. Cleavage kinetics may depend on the 'maturity' (solubility) of the collagen. The observation that UV-damaged mouse collagen is a better substrate for collagenase than the intact sample may be illustrative of a mechanism whereby damaged collagen targets itself for selective attack by collagenase.
Subject(s)
Collagen/metabolism , Collagen/radiation effects , Collagenases/metabolism , Ultraviolet Rays , Animals , Dose-Response Relationship, Radiation , Electrophoresis, Polyacrylamide Gel , Mice , Mice, HairlessABSTRACT
BACKGROUND: The sentinel lymph node (SLN) mapping technique has been used in breast cancer and melanoma, and was recently described for colon cancer. METHODS: Thirty-five patients with colon cancer underwent intraoperative SLN mapping. One milliliter of 1% isosulfan blue was injected subserosally around the tumor. The first nodal area that was highlighted with blue was identified as the SLN. All lymph nodes underwent examination with hematoxylin and eosin (H&E) stain. SLNs underwent additional sectioning and were stained with CAM 5.2. RESULTS: Lymphatic mapping adequately identified the SLN in 25 patients (71%). In the 15 cases where the SLNs were negative for metastases, all other non-SLNs were also negative (0% false negative rate). The SLN was the only site of metastases in 6 (17%) of 35 patients. CAM 5.2 staining provided the only evidence of micrometastases in 4 (11%) of 35 patients. CONCLUSIONS: Intraoperative SLN mapping is a feasible technique with a reasonable SLN identification rate (71%). The absence of metastases in the SLNs accurately predicts the status of the non-SLNs. Tumors in 11% of patients were upstaged by the demonstration of micrometastatic involvement, and these patients may benefit from further adjuvant chemotherapy.
Subject(s)
Adenocarcinoma/pathology , Colonic Neoplasms/pathology , Sentinel Lymph Node Biopsy/methods , Adenocarcinoma/surgery , Adult , Aged , Aged, 80 and over , Colonic Neoplasms/surgery , Female , Humans , Intraoperative Care , Male , Middle Aged , Prospective Studies , Rosaniline Dyes , Sensitivity and SpecificityABSTRACT
High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATA-binding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , TATA-Binding Protein Associated Factors , Transcription Factors/metabolism , DNA Footprinting , Drug Stability , Fungal Proteins/metabolism , Kinetics , Macromolecular Substances , Protein Binding , RNA Polymerase III/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins , TATA-Box Binding Protein , Thermodynamics , Transcription Factor TFIIIBABSTRACT
BACKGROUND: Collagens have the well-known ability to spontaneously self-associate to form fibrils at physiological temperature and neutral pH in vitro and in vivo. Because solar UV may photochemically alter collagen, the kinetics of fibril formation may be modified. Thus, we have begun a systematic study of the effect of various UV wavebands on fibril formation. METHODS: Citrate-soluble calf skin collagen (Elastin Products) was dissolved at 0.05% in 0.5 M HOAc, dialyzed over 2 days into two changes of 0.0327 M phosphate buffer, pH 7.0 at 4 degrees C, and centrifuged at 48,000 x g. Photolysis was carried out at 4 degrees C with either (a) UVC (UVG-11 lamp), (b) filtered solar-simulating radiation (SSR) or UVA (SSR or UVL-21 lamp filtered with a 2.0 mm Schott WG 345 filter). Gelation was commenced by rapidly raising the temperature from 8 degrees C to 33 degrees C. Nucleation and growth were followed by turbidimetric measurements at 400 nm. RESULTS: UVC radiation (0-17.3 J/cm2) resulted in a dose-dependent decrease in the rate of fibril growth. Under these conditions, concomitant collagen crosslinking and degradation occurred. Fibril nucleation, a prerequisite for growth, was rapid (threshold approximately 2 min) and was not affected by UVC, UVA or SSR. SSR (0-1,320 J/cm2) caused a small decrease in growth rate and in the degree of fibril formation. UVA radiation (0-1,080 J/cm2) had a similar effect. "Direct" photochemical damage thus paralleled absorption via various collagen chromophores, with UVC>SSR approximately UVA. The presence of riboflavin (RF) resulted in groundstate interactions that markedly altered both nucleation and growth kinetics. Irradiation with 29.6 J/ cm2 UVA in the presence of RF photosensitizer caused relatively minor additional changes in fibrillation kinetics. CONCLUSIONS: These results collectively indicate that fibril formation is markedly dependent on specific ground state interactions and relatively insensitive to nonspecific UV damage. On the other hand, fibrils thus formed from photochemically altered collagen may have altered structural properties that could have subtle but unfavorable effects on the local dermal milieu in vivo. Notwithstanding, the relative insensitivity of fibrillogenesis to non-specific photochemical damage probably represents a favorable adaptation, overall, which tends to conserve the mechanical integrity of the skin.
Subject(s)
Collagen Type I/biosynthesis , Animals , Buffers , Cattle , Dose-Response Relationship, Radiation , Hydrogen-Ion Concentration , Phosphates , Riboflavin , Ultraviolet RaysABSTRACT
Nhp6A and Nhp6B are HMG1-like proteins required for the growth of S. cerevisiae at elevated temperatures. We show that the conditional lethality of an nhp6 strain results from defective transcription of SNR6 (U6 snRNA) by RNA polymerase III. Overexpression of U6 snRNA or Brf1, a limiting component of TFIIIB, and an activating mutation (PCF1-1) in TFIIIC were each found to suppress the nhp6 growth defect. Additionally, U6 snRNA levels, which are reduced over 10-fold in nhp6 cells at 37 degrees C, were restored by Brf1 overexpression and by PCF1-1. Nhp6A protein specifically enhanced TFIIIC-dependent, but not TATA box-dependent, SNR6 transcription in vitro by facilitating TFIIIC binding to the SNR6 promoter. Thus, Nhp6 has a direct role in transcription complex assembly at SNR6.
Subject(s)
DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , High Mobility Group Proteins/metabolism , Nuclear Proteins/metabolism , RNA Polymerase III/metabolism , RNA, Small Nuclear/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Genes, Fungal/genetics , Genes, Lethal/genetics , HMGN Proteins , Nuclear Proteins/genetics , Phenotype , Promoter Regions, Genetic/genetics , Protein Binding , RNA Polymerase III/chemistry , RNA, Fungal/biosynthesis , RNA, Fungal/genetics , RNA, Ribosomal, 5S/biosynthesis , RNA, Ribosomal, 5S/genetics , RNA, Small Nuclear/metabolism , RNA, Transfer/biosynthesis , RNA, Transfer/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Suppression, Genetic/genetics , Temperature , Transcription Factor TFIIIB , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors, TFIII/genetics , Transcription Factors, TFIII/metabolism , Transcription, Genetic/geneticsABSTRACT
In the transcription of tRNA and 5 S genes by RNA polymerase III, recruitment of the transcription factor (TF)IIIB is mediated by the promoter-bound assembly factor TFIIIC. A critical limiting step in this process is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related factor Brf1p/TFIIIB70. To facilitate biochemical studies of this interaction, we expressed a fragment of TFIIIC131, TFIIIC131-(1-580), that includes the minimal TFIIIB70 interaction domain defined by two-hybrid studies together with adjacent sequences, up to the end of TPR9, implicated in the assembly reaction. TFIIIC131-(1-580) interacts with TFIIIB70 in solution and inhibits the formation of TFIIIB70.TFIIIC.DNA complexes. In a coupled equilibrium binding assay, the formation of TFIIIC131-(1-580).TFIIIB70 complexes was adequately described by a single-site binding model and yielded an apparent equilibrium dissociation constant of 334 +/- 23 nm. CD spectroscopy and limited proteolysis experiments defined a well structured and largely protease-resistant core in TFIIIC131-(1-580) comprising part of the hydrophilic amino terminus, TPR1-5, the intervening non-TPR region, and TPR6-8. CD spectra showed that trifluoroethanol induced significant alpha-helical structure in TFIIIC131-(1-580). A more modest monovalent ion-dependent CD difference was observed in mixtures of TFIIIC131-(1-580) and TFIIIB70, suggesting that formation of the binary complex may proceed with the acquisition of alpha-helicity.
Subject(s)
Acetyltransferases/metabolism , Transcription Factors, TFIII/metabolism , Transcription Factors/metabolism , Circular Dichroism , DNA Polymerase III/metabolism , Fungal Proteins/chemistry , Peptide Fragments/chemistry , Protein Binding , Protein Conformation , Transcription Factor TFIIIB , Transcription, Genetic , YeastsABSTRACT
The transcription of ribosomal DNA, ribosomal protein (RP) genes, and 5S and tRNA genes by RNA polymerases (Pols) I, II, and III, respectively, is rapidly and coordinately repressed upon interruption of the secretory pathway in Saccharomyces cerevisiae. We find that repression of ribosome and tRNA synthesis in secretion-defective cells involves activation of the cell integrity pathway. Transcriptional repression requires the upstream components of this pathway, including the Wsc family of putative plasma membrane sensors and protein kinase C (PKC), but not the downstream Bck1-Mkk1/2-Slt2 mitogen-activated protein kinase cascade. These findings reveal a novel PKC effector pathway that controls more than 85% of nuclear transcription. It is proposed that the coordination of ribosome and tRNA synthesis with cell growth may be achieved, in part, by monitoring the turgor pressure of the cell.
Subject(s)
RNA, Transfer/biosynthesis , Ribosomes/metabolism , Signal Transduction , DNA-Binding Proteins/metabolism , Protein Kinase C/metabolism , RNA Polymerase II/metabolism , RNA Polymerase III/metabolism , RNA, Transfer/genetics , Ribosomal Proteins/geneticsABSTRACT
We report herein a unique case of an esophageal collision tumor composed of a papillary adenocarcinoma and a large cell neuroendocrine carcinoma arising in a Barrett esophagus. Hematoxylin-eosin and silver staining patterns, immunohistochemistry, and electron microscopy of the large cell neuroendocrine component are discussed.
Subject(s)
Adenocarcinoma, Papillary/pathology , Barrett Esophagus/pathology , Carcinoma, Neuroendocrine/pathology , Esophageal Neoplasms/pathology , Neoplasms, Second Primary/pathology , Adenocarcinoma, Papillary/chemistry , Adenocarcinoma, Papillary/surgery , Barrett Esophagus/metabolism , Barrett Esophagus/surgery , Biomarkers, Tumor/analysis , Carcinoma, Neuroendocrine/chemistry , Carcinoma, Neuroendocrine/surgery , Esophageal Neoplasms/chemistry , Esophageal Neoplasms/surgery , Humans , Immunoenzyme Techniques , Male , Middle Aged , Neoplasm Proteins/analysis , Neoplasms, Second Primary/chemistry , Neoplasms, Second Primary/surgeryABSTRACT
A case of clear-cell epithelioid leiomyoma of the round ligament in a 69-year-old woman is described. The neoplasm presented as a firm left inguinal mass. A preoperative computed tomography (CT) scan demonstrated an enhancing lesion extending extra-abdominally from the region of the external inguinal ring. The surgical resection specimen was tan-white, well-circumscribed, and measured 5.8 cm in maximum dimension. Microscopic examination revealed a well-demarcated neoplasm comprised of polygonal cells with abundant clear cytoplasm arranged in clusters and single files with abundant intervening hyalinized stroma. There was minimal nuclear atypia and mitotic figures were rare. Periodic acid-Schiff with diastase demonstrated intracytoplasmic glycogen. Immunohistochemical stains for pan-actin, smooth muscle actin, desmin, bcl-2, and vimentin were positive in the tumor cells, whereas stains for CD34, carcinoembryonic antigen, cytokeratin, epithelial membrane antigen, S100 protein, and neurofilaments were negative. Electron microscopy demonstrated features of smooth muscle differentiation including longitudinally oriented fine filaments with focal condensations, pinocytotic activity, and subplasmalemmal densities. This case illustrates the ubiquitous distribution of epithelioid smooth muscle neoplasms and highlights the potential pitfalls for diagnosis when they occur in an unusual location.
Subject(s)
Genital Neoplasms, Female/pathology , Leiomyoma, Epithelioid/pathology , Round Ligament of Uterus , Actins/analysis , Aged , Desmin/analysis , Female , Genital Neoplasms, Female/metabolism , Genital Neoplasms, Female/ultrastructure , Humans , Immunohistochemistry , Leiomyoma, Epithelioid/metabolism , Leiomyoma, Epithelioid/ultrastructure , Muscle, Smooth/metabolism , Proto-Oncogene Proteins c-bcl-2/analysis , Tomography, X-Ray Computed , Vimentin/analysisABSTRACT
We report a case of predominantly intraductal carcinoma of the pancreas with microscopic foci of invasive carcinoma in a patient with chronic pancreatitis. In this article, we discuss the pathologic and prognostic features of pancreatic carcinoma in situ. This entity is probably overlooked due to a number of reasons, including the fact that, in most cases, pancreatic ductal carcinomas are extensively infiltrative at the time of surgical removal; the atypical epithelial changes in the intraductal carcinoma had been overlooked in the presence or absence of an invasive component; epithelial changes may be missed due to insufficient sampling; and last, the differentiation with atypical epithelial hyperplasia is a subjective matter. Intraductal carcinoma of the pancreas is a distinct pathological entity with characteristic morphologic changes restricted to the ductal epithelium, bearing important prognostic implications.
Subject(s)
Carcinoma in Situ/pathology , Pancreatic Neoplasms/pathology , Aged , Animals , Carcinoma in Situ/complications , Cholangiopancreatography, Endoscopic Retrograde , Chronic Disease , Disease Progression , Female , Humans , Hyperplasia/pathology , Neoplasm Invasiveness , Pancreatic Ducts/pathology , Pancreatic Neoplasms/complications , Pancreatitis/complicationsABSTRACT
"Special" highly protective fabrics are now available that offer broad-spectrum protection in preventing sunburn, and possibly other types of photodamage. It is important to know to what extent these fabrics are capable of protecting the wearer against skin cancer, photosensitivity disorders, and inadvertent phototoxic reactions from photodynamic therapy (PDT). We assess the ability of one such special (Solumbra) fabric and one "typical" summer fabric to provide protection against PDT phototoxicity produced in tape-stripped Sk-1 hairless mice by topical 5-aminolevulinic acid (ALA) and (primarily) visible light (360-800 nm). Since ALA-derived photosensitizers absorb most of the visible spectrum, results from these studies give a good indication of the photoprotective capability of these fabrics throughout this region. Mice were irradiated dorsally with a Kodak slide projector equipped with a 300 W tungsten-halogen lamp (I0 = 48.3 mW/cm2). After determining the minimal phototoxic dose (MPD) to be 1.40 +/- 0.4 J/cm2, we irradiated the tape-stripped ALA-sensitized mice through the stretched test fabrics with appropriate multiples of the MPD. The special fabric provided protection against 25-30 MPD visible light between 360-800 nm in 14/14 mice. The typical fabric failed to provide protection against 2.5 MPD of the same spectrum. No phototoxic or other adverse responses were seen with either the ALA or light control. In conclusion, the Solumbra fabric is much more protective against ALA photosensitization than the typical fabric. Both appear better at blocking UV than visible light.
Subject(s)
Photochemotherapy , Photosensitivity Disorders/prevention & control , Radiation Protection , Skin/radiation effects , Textiles , Aminolevulinic Acid/administration & dosage , Animals , Mice , Mice, Hairless , Photosensitizing Agents/administration & dosage , Skin/pathology , Ultraviolet Rays/adverse effectsABSTRACT
Several important clinical conditions can result in close association between the pigment melanin and dermal collagen. Because melanin and its precursors can be chemically reactive in ground and excited states, it is important to know whether the resulting melanin-collagen interaction results in photoprotection or photoaggression. Acidic and neutral air-saturated collagen suspensions (0.033%) were irradiated with 0-2.6 x 10(4) J/m2 UVC or with 0-83 x 10(4) J/m2 solar-simulating UV radiation (SSR). Photochemical destruction of a photolabile collagen fluorophore (lambda em 360 nm) and collagen chain degradation were monitored as functions of irradiation time in the presence and absence of added (0-100 micrograms) sepia eumelanin. Melanin retarded collagen photodamage but did not qualitatively alter the fluorescence fading kinetics. Both H2O2 and O2-. can be produced by UV irradiation of eumelanin. Added H2O2 and KO2 destroyed collagen fluorescence and caused 50% chain degradation at ca 10-20-fold molar excess. Previous studies have demonstrated that eumelanins efficiently scavenge O2-.. We demonstrated that eumelanin also efficiently scavenges H2O2 as evidenced by its ability to (a) compete with scopoletin for peroxide uptake and (b) directly take up H2O2 through a dialysis bag. The latter observation suggests that peroxide scavenging could occur in vivo by melanin sequestered in melanophages. Thus, neither UV-generated O2-. nor H2O2 are likely to be present in concentrations high enough to cause measurable collagen damage. Absorption and/or scattering of excitation radiation away from the target chromophore appears to be the primary photoprotection mechanism, although scavenging of active O2 intermediates may play an important, if subtle role.
Subject(s)
Collagen/radiation effects , Melanins/pharmacology , Radiation-Protective Agents/pharmacology , Sunlight , Ultraviolet Rays , Animals , Collagen/drug effects , Free Radical Scavengers/pharmacology , Hydrogen Peroxide/pharmacology , Kinetics , Melanins/isolation & purification , Mollusca , PhotolysisABSTRACT
TFIIIB, the initiation factor for transcription by RNA polymerase III (pol III) is, in yeast, composed of three subunits: TBP, TFIIIB70/Brf1 and TFIIIB90. To determine the extent to which each of these subunits is limiting for pol III transcription, the effect of overexpressing each subunit was assessed on the expression of wild-type and promoter mutant pol III genes both in vivo and in vitro . In vivo , we find that the synthesis of wild-type pol III genes is not limited to a significant extent by the level of any TFIIIB subunit. There is, however, a two-fold increase in the synthesis of the promoter mutant gene, sup9-e A19-supS1 , in strains overexpressing TFIIIB70. The findings suggest that overexpression of TFIIIB70has a differential effect on the expression of pol III genes with strong versus weak promoters. In vitro transcription assays support this conclusion and reveal an inverse correlation between the transcriptional response to TFIIIB70overexpression and promoter strength. The individual TFIIIB subunits are nuclear by immunofluorescence and are calculated to have nuclear concentrations in the low micromolar range. In comparison, the factors are diluted 100-fold or more in whole cell extracts. This dilution accounts for the generally limiting nature of TFIIIB70in pol III gene transcription in vitro.
Subject(s)
Gene Expression Regulation, Fungal/genetics , Promoter Regions, Genetic/genetics , RNA Polymerase III/metabolism , Saccharomyces cerevisiae/genetics , Transcription Factors/metabolism , Cell Nucleus/chemistry , DNA-Binding Proteins/analysis , Mutation , RNA Precursors/biosynthesis , RNA, Fungal/biosynthesis , Suppression, Genetic , TATA-Box Binding Protein , Transcription Factor TFIIB , Transcription Factors/analysis , Transcription Factors/geneticsABSTRACT
We have conducted a quantitative thermodynamic study of the effects of the TATA element and TATA-flanking sequences on the assembly of complexes containing TATA-binding protein (TBP) and the TFIIB-related factor, TFIIIB70. TBP binds to the sequence TATAAAAG in the context of the yeast U6 gene (yU6 hybrid TATA) or the adenovirus major late promoter (AdMLP) with different affinities demonstrating that the sequence context of a TATA element contributes to TBP binding. We also determined the cooperative free energies of formation of TBP.TFIIIB70.DNA complexes on the yU6 TATA element, the yU6 hybrid TATA element and a nonconsensus TATA element. The yU6 hybrid TATA displayed a moderate, less than 5-fold, increase in TBP affinity similar to the 3-fold increase observed for the AdMLP. In contrast, the nonconsensus and yU6 TATAs increased the affinity of TBP for DNA 12- and 17-fold, respectively. Since the TBP-TFIIIB70 cooperativity is greater on lower affinity TATA boxes and most polymerase III genes contain low affinity "TATA boxes," we conclude that the cooperative binding of TFIIIB70 and TBP to DNA represents an important driving force in the assembly of polymerase III-specific transcription complexes. An effect of the sequences surrounding the TATA box was also observed on TBP-TFIIIB70 cooperativity. The mechanistic implications of the thermodynamic linkage between DNA sequence and binding cooperativity are discussed.
Subject(s)
DNA-Binding Proteins/metabolism , TATA Box , Transcription Factors/metabolism , Adenoviridae/genetics , Base Sequence , DNA, Viral , DNA-Binding Proteins/chemistry , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , TATA-Box Binding Protein , Thermodynamics , Transcription Factor TFIIIB , Transcription Factors/chemistryABSTRACT
Transcription factor IIIC (TFIIIC) plays an important role in assembling the initiation factor TFIIIB on genes transcribed by RNA polymerase III (Pol III). In Saccharomyces cerevisiae, assembly of the TFIIIB complex by promoter-bound TFIIIC is thought to be initiated by its tetratricopeptide repeat (TPR)-containing subunit, TFIIIC131, which interacts directly with the TFIIB-related factor, TFIIIB70/Brf1. In this work, we have identified 10 dominant mutations in TFIIIC131 that increase Pol III gene transcription. All of these mutations are found within a discrete 53-amino-acid region of the protein encompassing TPR2. Biochemical studies of one of the mutations (PCF1-2) show that the increase in transcription is due to an increase in the recruitment of TFIIIB70 to TFIIC-DNA. The PCF1-2 mutation does not affect the affinity of TFIIIC for DNA, and the differential in both transcription and TFIIIB complex assembly is observed at saturating levels of TFIIIB70. This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB70 and suggests that the increased recruitment of this factor is achieved by a nonequilibrium binding mechanism. A novel mechanism of activation in which the TPR mutations facilitate a conformational change in TFIIIC that is required for TFIIIB70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.
Subject(s)
Mutation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transcription Factors, TFIII , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Binding Sites/genetics , DNA, Fungal/genetics , DNA, Fungal/metabolism , Models, Biological , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid , Transcription Factor TFIIB , Transcription Factor TFIIIB , Transcription Factors/chemistry , Transcription, GeneticABSTRACT
The polyquinoid nature of eumelanin(s) enables them to couple oxidation of electron donors with the reduction of electron acceptors. We have studied the ability of synthetic (Sigma) and "biological" (cuttlefish sepia) melanins to mediate electron transfer between hydroxybenzene donors (tyrosine, dopa, chemical depigmenters) and model acceptors (ferricyanide, tyrosinase). 1) Depending on the reductant, melanin either retards or accelerates ferricyanide reduction. Reaction kinetics are consistent with a mechanism involving non-interactive binding of both hydroxybenzene and ferricyanide to melanin prior to coupled electron transfer. 2) Melanins also act as an electron conduit in markedly accelerating the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH). The active species appears to be a complex between melanin and MMEH. The magnitude of both effects depend on the type of melanin as well as its oxidation state. Sepia (eu)melanin appears to protect against UV-induced damage to acid-soluble collagen, as judged by irreversible loss of intrinsic collagen fluorescence. Photoprotection against this type of damage appears primarily to involve optical absorption/scattering by the pigment.
Subject(s)
Melanins/chemistry , Radiation Tolerance/physiology , Collagen/radiation effects , Electron Transport , Ferricyanides/metabolism , Melanins/physiology , Oxidation-Reduction , Radiation-Protective Agents , Solutions , Ultraviolet RaysABSTRACT
BACKGROUND: Butenafine hydrochloride, a benzylamine derivative with potent antifungal activity, has been used in Japan to treat superficial fungal diseases. OBJECTIVE: We evaluated the safety and efficacy of twice-daily butenafine versus its vehicle in the treatment of interdigital tinea pedis in a multicenter, randomized, double-blind, parallel-group trial. METHODS: A total of 402 patients with interdigital tinea pedis and a positive potassium hydroxide examination were enrolled. Of the 271 patients who had culture-confirmed tinea pedis and were assessed for efficacy, 132 applied butenafine and 139 applied vehicle twice daily for 1 week. Patients were assessed for mycologic cure, effective treatment, overall cure, and mycologic/clinical cure. RESULTS: The rates of all four end points were significantly higher with butenafine than with vehicle 5 weeks after treatment ended. Rates of mycologic cure and effective treatment with butenafine were significantly higher than with vehicle at cessation of treatment. Adverse events to treatment occurred in less than 1% of patients treated with butenafine and 2% of patients who applied vehicle. CONCLUSION: Butenafine applied twice daily for 1 week is highly effective in treating interdigital tinea pedis.
Subject(s)
Antifungal Agents/administration & dosage , Benzylamines/administration & dosage , Naphthalenes/administration & dosage , Tinea Pedis/drug therapy , Administration, Topical , Adolescent , Adult , Aged , Aged, 80 and over , Analysis of Variance , Antifungal Agents/adverse effects , Benzylamines/adverse effects , Double-Blind Method , Drug Administration Schedule , Female , Follow-Up Studies , Humans , Male , Middle Aged , Naphthalenes/adverse effects , Recurrence , Treatment OutcomeABSTRACT
Transcription by RNA polymerase III (pol III) in yeast requires the assembly of an initiation complex comprising the TATA-binding protein (TBP), a 90-kDa polypeptide (TFIIIB90), and a 70-kDa polypeptide (TFIIIB70). TFIIIB70 interacts with TBP, a unique pol III subunit, C34, and the 131-kDa subunit of the pol III-specific complex, TFIIIC. TFIIIB70 was expressed in Escherichia coli and purified to homogeneity. The specific transcription activity of rTFIIIB70 is 22-58% that of the native yeast and in vitro synthesized factor. However, only a small fraction (0.07-0.32%) of the TFIIIB70 from these sources results in the synthesis of full-length RNA. The data suggest that TFIIIB70 function may be limited by an unfavorable recruitment equilibrium into the preinitiation complex. Quantitative DNase I "footprint" titrations of yeast TBP to the adenovirus major late promoter were conducted at a series of constant TFIIIB70 concentrations. A value of -0.7 +/- 0.2 kcal/mol was determined for the cooperative free energy of formation of the TBP.TFIIIB70.DNA complex at concentrations of TFIIIB70 sufficient to partition all of the binding cooperativity to the TBP binding isotherm. A Kd of 44 +/- 23 nM characterizes the TFIIIB70 concentration dependence of the TBP.TFIIIB70 cooperativity. The relationship deltalog K/deltalog (TFIIIB70) is consistent with the linkage of a single molecule of TFIIIB70 with the TBP-promoter binding reaction.