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PubMed1987

Rapid sample preparation methods for analysis of residues of sulfamethazine and its N4-acetyl and desamino metabolites in swine tissue by HPLC.

Rapid sample preparation methods for the determination of sulfamethazine (SMZ) and its N4-acetyl and desamino metabolites in swine tissues at the 0.1 mg kg-1 level are presented. The methods use sonication-aided extraction with dichloromethane. For SMZ and N4-acetyl SMZ analysis extracts are cleaned up and concentrated on a silica disposable column followed by HPLC on a CP Spher C8 column using acetonitrile-sodium acetate buffer as the mobile phase. For desamino-SMZ analysis the extract was cleaned up and concentrated on a Florisil disposable column, followed by HPLC on a Nucleosil 5-CN column after formation of an ion-pair complex with 1-heptanesulfonic acid. For desamino SMZ peak identification by diode-array UV/VIS detection is also described. Mean recoveries from spiked tissue samples were about 87% (muscle) and 76% (kidney) for SMZ and N4-acetyl SMZ and about 70% for desamino SMZ.

Animals↗

PubMed1990

Spatial differences of endogenous lectin expression within the cellular organization of the human heart: a glycohistochemical, immunohistochemical, and glycobiochemical study.

Protein-carbohydrate recognition may be involved in an array of molecular interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, surgically removed specimens of human endomyocardial tissue were processed for histochemical and biochemical analysis. The inherent capacity of these sections to bind individual sugar moieties, which are constituents of the carbohydrate part of cellular glycoconjugates, was assessed using a panel of biotinylated neoglycoproteins according to a standardized procedure. Together with appropriate controls, it primarily allowed localization of endogenous lectins. Differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures. The endocardium proved to be positive with beta-galactoside-bearing probes; with neoglycoproteins carrying beta-xylosides, alpha-fucosides, and galactose-6-phosphate moieties; and with probes containing a carboxyl group within the carbohydrate structure, namely sialic acid and glucuronic acid. In contrast, only fucose-and maltose-specific receptors were apparent in the elastic layers of the endocardium. Aside from ascertaining the specificity of the protein-carbohydrate interaction by controls, i.e., lack of binding of the probe in the presence of the unlabelled neoglycoprotein and lack of binding of the labelled sugar-free carrier protein, respective sugar receptors were isolated from heart extracts by using histochemically effective carbohydrates as immobilized affinity ligand. Moreover, affinity chromatography using immobilized lactose as affinity ligand as well as the use of polyclonal antibodies against the predominant beta-galactoside-specific lectin of heart demonstrated that the lactose-specific neoglycoprotein binding was due to this lectin. Remarkably, the labelled endogenous lectin, preferred to plant lectins for detecting ligands of the endogenous lectin, localized ligands in tissue parts where the lectin itself was detected glycohistochemically as well as immunohistologically. This demonstration of receptor-ligand presence in the same system is a further step toward functional assignment of the recorded protein-carbohydrate interaction. Overall, the observed patterns of lectin expression may serve as a guideline to elucidate the precise physiological relevance of lectins and to analyze pathological conditions comparatively.

Endocardium↗

PubMed1991

Alterations in human lung parenchyma after cytostatic therapy.

Chemotherapy does not only affect the viability of the tumor cell. It may also cause alterations in normal organs. Thus, tumor-free areas within human lung parenchyma of 63 surgical specimens of intrapulmonary metastases were analyzed to assess the extent of morphologic changes in response to previous cytostatic therapy. The material included 34 cases of sarcoma, 20 cases of germ cell tumors, 6 cases of hypernephroid carcinoma, two cases of mammary carcinoma and one case of metastatic melanoma. All patients had received cytostatic therapy in generally applied regimens for more than two years. Morphologic analysis was carried out by routine procedures. In addition to conventional staining procedures including HE, PAS, and Sirius stain, further tools were employed to extend the array of determined characteristics. To evaluate any changes in the tissue in order to specifically recognized carbohydrate structures, labeled neoglycoproteins or proteoglycans with specificity for endogenous receptors that bind to mannose, maltose, L-fucose, lactose, N-acetyl-D-glucosamine, and heparin were used. A monoclonal antibody binding the HLA-DR receptor was also included in the study. As a control, sections of 20 cases with intrapulmonary metastases without exposure to previous cytostatic therapy were included. To address the further question whether cytostatic therapy may induces changes in tumor-free lung that show similarities to the organ in question, sections from 18 cases with tuberculosis and from 37 cases suffering from sarcoidosis were similarly examined. Focal interstitial fibrosis was seen in 28/63 (44%) of the patients receiving chemotherapy. In contrast, only 2/20 (10%) patients of the untreated group exhibited this alteration. An active fibrosis with proliferating smooth muscle cells was found in two cases, dysplastic pneumocytes in 10 cases (16%) in the group with cytostatic therapy, but in no cases in the untreated group. Expression of the HLA-DR receptor in the pneumocytes was observed in 27/63 cases (43%) of the cytostatic cohort, in 21/37 (57%) patients of the sarcoidosis cohort, in 15/18 (83%) patients of the tuberculosis cohort, and in 1/20 (5%) of the untreated patients. In contrast to sections from treated patients, binding of neoglycoproteins was low in the untreated cohort. Interestingly, similarities between the tuberculosis cohort and the cytostatic cohort were seen for receptors that are specific for fucose and lactose, respectively. The results suggest that long-lasting cytostatic therapy induces focal fibrosis in 40%-50% of the patients, mainly via unspecific interstitial inflammatory infiltrates. A hypersensitivity reaction or direct toxicity may less frequently lead to pathologic alterations.

Adult↗

PubMed1989

The development of microtubular arrays in the germ tissue of an insect telotrophic ovary.

The microtubular arrays characteristic of the trophic core and cords of the adult Rhodnius prolixus ovary develop prior and during the larval--adult transformation. Development of the microtubules was revealed by immunocytochemistry, electron microscopy and polyacrylamide electrophoresis and Western blot analysis. Microtubular arrays were first detected in the trophic cords and presumptive trophic core 6 days before the adult molt. Cord microtubules increase in length and numbers as the trophic cords grow. Three microtubule packed cords have formed by 1 day post molt. The microtubule distribution in the presumptive core is non-uniform. Microtubule packed areas are interspersed with areas devoid of microtubules. The adult core begins forming between 1 day before molt and molting. This early adult core arises from the fusion of the anterior portions of microtubule packed cords. A fully mature adult core is not present by 2 days post molt. The microtubule packing density in the core increases from 2 days before to 2 days post molt. Tubulin increases from 6 days to 1 day before the adult molt.

Animals↗

PubMed2024

PANAMA-enabled high-sensitivity dual nanoflow LC-MS metabolomics and proteomics analysis.

High-sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient at preventing nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomics (including lipidomics) and proteomics from the same samples without instrument duplication. This workflow is based on a robust solid-phase micro-extraction step for routine sample cleanup and bioactive molecule enrichment. Our method, termed proteomic and nanoflow metabolomic analysis (PANAMA), improves compound resolution and detection sensitivity without compromising the depth of coverage as compared with existing widely used analytical procedures. Notably, PANAMA can be applied to a broad array of specimens, including biofluids, cell lines, and tissue samples. It generates high-quality, information-rich metabolite-protein datasets while bypassing the need for specialized instrumentation.

Proteomics↗

PubMed1987

Heating deep seated eccentrically located tumors with an annular phased array system: a comparative clinical study using two annular array operating configurations.

Regional heating administered with an annular array to 12 patients with deep-seated advanced malignant disease eccentrically located in the lower abdomen and pelvis is compared based on the annular array operating configuration. One configuration (4 quadrants active) delivers radiofrequency power with relative uniformity throughout the patient cross-section. The other (2 quadrants active) allows the radiofrequency power deposition to be shifted preferentially into the eccentrically located treatment volume. Phantom measurements have been made to demonstrate the redistribution of radiofrequency power that results when the annular array is operated in these respective configurations. Systemic responses (i.e. oral temperature rise, changes in blood pressure, and heart rate) to these regional hyperthermia applications are compared and are not significantly different with respect to these heating configurations. Temperature data obtained during treatment sessions using these two annular array operating configurations are analyzed based on the fraction of measured tumor and normal tissue temperatures exceeding or equal to a given index temperature. Although the two quadrant configuration is more efficient in delivering power to the treatment volume, this analysis does not indicate a significant gain in therapeutic heating as a result of this preferential power deposition. Treatment tolerance and heterogeneity with respect to tissue type and blood flow remained the dominant limiting factors with regard to temperatures achieved.

Abdominal Neoplasms↗

PubMed1985

Primary culture of duck salt gland. I. Morphology of confluent cell layers.

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.

Animals↗

PubMed1987

Morphometry of bladder carcinoma: I. The automatic delineation of urothelial nuclei in tissue sections using an IBAS II image array processor.

Semi-automatic methods of morphometry require the boundaries of the objects under study to be drawn by hand. This is slow, tedious and does not guarantee accuracy. Complete automation, with recognition, detection and verification by computer is not yet feasible. Automatic detection of nuclear boundaries with observer verification provides a means of obtaining large amounts of data for analysis, quickly and with similar accuracy to manual tracing.

Cell Nucleus↗

PubMed1987

Photodynamic therapy in the normal rat colon with phthalocyanine sensitisation.

Photodynamic therapy (PDT) involves the interaction of light with an administered photosensitising agent to produce cellular destruction. It has promising potential for the local and endoscopic treatment of gastrointestinal cancer. There is however little data on the response of normal intestine to PDT. We have investigated the use of a new photosensitiser chloro aluminum sulphonated phthalocyanine (AlSPc) for colonic PDT. The peak concentration of AlSPc in the colon measured by alkali extraction occurred 1 h after i.v. injection. The cellular uptake demonstrated by laser fluorescence microscopy was greater in the mucosa than in the muscle. AlSPc was activated in the tissues by light from an argon ion pumped dye laser at 675 nm. The laser power was set at 100 mW and the fibre placed touching the mucosa. In control animals no macroscopic damage was seen. Temperature measurement using a microthermocouple array showed no temperature rise during light exposure. The energy (fluence), dose of sensitiser and time from sensitisation to phototherapy were altered and the area of necrosis measured. The geometry of the colon made theoretical analysis of the correlation between laser energy and size of lesion difficult. However, following direct measurement of the relative light intensity (fluence rate) in the colon we were able to confirm that there was a threshold fluence for colonic necrosis. The area of photodynamic damage seen 72 h after phototherapy fell with the fall in tissue concentration of AlSPc from 1 h to 1 month after i.v. injection. However, maximum tissue necrosis occurred when treatment was performed immediately after i.v. injection. In this situation, intense vascular spasm was seen and any light transmitted through the colon which fell on the small bowel mesentery caused a lethal ischaemic necrosis. The initial histological changes after PDT were vascular, followed by full thickness necrosis at 72 h. Healing by regeneration was complete by 2-3 weeks. Despite full thickness necrosis there was no reduction in the colonic bursting pressure at any time. Colon treated by hyperthermia had a reduced bursting pressure. Specific collagen stains showed that PDT did not alter the submucosal collagen architecture whereas hyperthermia did.

Animals↗

PubMedDate not supplied

Cerebral distribution of 133-Xe and blood flow measured with high purity germanium.

Distribution of cerebral blood flow was measured with an array of 200 ultra-pure germanium radiation detectors and 133-Xe by inhalation. The array "sees" the head as a composite of different subvolumes and enables measurement of the concentration history of tracer every 1-10 sec in each subvolume simultaneously. Subvolume mean flows, (fm), and partition coefficients, lambda m, are derived by compartmental analysis of tissue concentration washout curves. Errors from "cross talk," scalp radiation, "look through," and assumed partition coefficients are eliminated. Average fm adjusted for 40 mm Hg PACO2 in 14 cortical subvolumes (7 right, 7 left) of four normal 21-24 year old controls ranged from 50 to 60 ml/100 cc tissue/min, and lambda m ranged from 0.97 to 1.14. Average fm and lambda m in white matter was 24 ml/100 cc/min and 1.42 - 1.14 respectively. During CO2 inhalation, right and left hemispheric fm increased 6.4% and 5.7%/mm Hg respectively, whereas white matter fm increased 2.2% and 3.4% mm Hg respectively. There was no systematic difference between front and back or dominant vs non-dominant sides. Three 73-84 year old controls had reduced fm and CO2 reactivity in all subvolumes, lambda m was in the same range as in younger controls. Two patients with intracranial cerebrovascular disease showed excellent localization of ischemic subvolumes. One patient with asymptomatic unilateral 98% stenosis of the internal carotid artery had a similar distribution of blood flow in both hemispheres.

Adult↗

PubMedDate not supplied

Potential for localized, adjustable deep heating in soft-tissue environments with a 30-beam ultrasonic hyperthermia system.

Initial heating rates (degrees C/min) along parallel tracks at depths of 1-14 cm in a static, muscle-like phantom were determined from time-temperature profiles obtained with 'Helios', a 30-beam ultrasonic hyperthermia system developed by Varian Associates. Data were taken at a single operating frequency of 556 kHz, for different sets of focal plane ring diameters of the four-ring array applicator, different levels of transducer driving power and two different focal plane depths, 6 cm and 9 cm. In each experiment, at each point of temperature measurement, analysis of temperature versus time data over a 2 min heating interval permitted separation of the desired phantom heating from artefactual heating resulting primarily from absorption of transverse (shear) waves produced at phantom-metal probe catheter interfaces. The results of the studies conducted suggest that in a non-translating carriage mode, Helios can produce axially and laterally localized deep heating in soft tissues for tissue volumes of lateral dimension up to a minimum of 4 cm and tissue depths of at least 11 cm. The results obtained also suggest that Helios can produce laterally localized heating to tissue depths of at least 11 cm without excessive heating of superficial soft-tissue layers, for tissue volumes of lateral dimension up to a minimum of 8 cm. The methodology used in the phantom studies was applied to the production of localized heating in the right lobe of the liver of adult pigs. Temperature versus time profiles obtained in the in vivo studies indicated that, for the set of system parameters employed, concentration of ultrasonic power at greater depths in the liver (e.g. 10.5 cm versus 5 cm) could be achieved, suggesting that Helios should be able to produce localized heating of targeted hepatic volumes when its operating parameters are selected in accordance with effective treatment planning techniques.

Animals↗

PubMedDate not supplied

Characterization of age-associated kidney disease in Wistar rats.

Kidney disease was studied patho-histologically, electron microscopically and immunologically in Wistar rats ranging in age from 2 weeks to 24 months. Glomerular lesions characterized by adhesion of podocyte foot processes were first detected at 3 months of age. The kidney lesions became more pronounced with age, as manifested by an increase of mesangial matrix, basement membrane thickening, crescent formation and hyalinization of glomeruli, and tubular degeneration. Evidence for the deposition of immune complexes in the kidney was obtained by immunofluorescence and electron microscopy, as well as by immuno-electrophoretic analysis of kidney elutions. Tests with antiserum reagent against Moloney leukemia virus antigen revealed its presence in some but not all glomeruli. The presence of other viruses in Moloney leukemia virus negative glomeruli was not ruled out. Serum autoantibody against an array of rat tissues could not be detected. Therefore, it would appear that autoimmune mechanism may not be the primary underlying cause of pathogenesis of the disease. Accordingly, the disease could be referred to as chronic immune complex glomerulonephropathy with nephrotic syndrome, but sources of the antigens in the complex were mostly unknown, althougn virus could be one portion of them. The possibility that diet antigens may also be present in the complex seems unlikely because attempts to demonstrate serum antibodies against diet pellet in old rats were unsuccessful.

Aging↗

PubMed2026

Beckwith-Wiedemann spectrum exhibiting a 46,XY karyotype caused by genome-wide paternal uniparental heterodisomy: a case report.

BACKGROUNDS: Patients with genome-wide paternal uniparental disomy (GWpUPD) usually exhibit clinical features of Beckwith-Wiedemann syndrome (BWS) and a 46,XX karyotype, with all chromosomes showing isodisomy. To date, male patients with GWpUPD and a complete 46,XY karyotype, specifically involving heterodisomy, have not been described. RESULTS: We report a male infant exhibiting classical BWS clinical features. DNA methylation analyses showed paternal-specific methylation across multiple imprinted loci, suggesting GWpUPD. Genetic analysis of autosomes and sex chromosomes indicated two distinct paternal genomes in peripheral blood leukocytes, whereas a normal biparental genome was detected in other tissues under chimeric conditions. These findings indicated that the patient had genome-wide paternal uniparental heterodisomy (GWpUPhD). The SNP array revealed the presence of one copy of the X chromosome and one copy of the Y chromosome, the patient is a chimera composed of 46,XY biparental cells (with maternal X) and 46,XY GWpUPhD cells (with paternal X). CONCLUSIONS: This is the first report of a male patient with a GWpUPhD chimera. We propose a potential mechanism of GWpUPhD formation. Our findings expand the molecular spectrum of GWpUPD and provide valuable insights into its pathogenesis in chimeric conditions. Furthermore, the potential for clinical manifestations unique to 46,XY heterodisomy warrants careful long-term follow-up.

Humans↗

PubMed1985

High-performance liquid chromatographic strategies for the determination and confirmation of anticoagulant rodenticide residues in animal tissues.

A comprehensive approach to the analysis of anticoagulant rodenticide residues in animal tissues based on high-performance liquid chromatography (HPLC) has been developed. Residues of warfarin, coumatetralyl, difenacoum, brodifacoum, bromadiolone, diphacinone and chlorophacinone were extracted with chloroformacetone (1:1, v/v). Extracts were cleaned-up by an integrated gel permeation and adsorption chromatographic procedure which divided the rodenticides into two groups. Residues were then determined and confirmed using normal-phase, ion-pair and weak ion-exchange HPLC techniques. Ion-pair gradient separation resolved all seven rodenticides in a single chromatographic analysis. UV detection methods were employed for all seven rodenticides. Use of a diode array detection system permitted additional confirmation of residues down to 0.1 mg kg-1 by matching UV spectra and derivatives of spectra. Sensitive fluorescence detection was possible for the coumarin-based rodenticides but not for diphacinone and chlorophacinone. Post-column pH-switching fluorescence detection methods were shown to be superior to other methods of fluorescence detection of coumarin-based rodenticides. Recoveries from spiked liver tissue were around 90% at levels from 0.05 to 1 mg kg-1. Detection limits of around 0.002 mg kg-1 for most rodenticides and of 0.01 mg kg-1 for warfarin could be achieved with animal tissue extracts.

Animals↗

PubMed1991

Primary structure of the human laminin A chain. Limited expression in human tissues.

cDNA clones for the human laminin A chain were isolated from libraries prepared from human gestational choriocarcinoma cell line (JAR) RNA. They cover approx. 8 kb from the 5'-end of the 9.5 kb mRNA coding for this protein. Our clones contain 94 nucleotide residues for the 5'-end untranslated region and 7885 nucleotide residues of coding sequence. The complete human laminin A chain contains a 17-amino acid-residue signal peptide and a 3058-residue A chain proper. The human laminin A chain has a distinct domain structure with numerous internal cysteine-rich repeats. The large globular domain G has five repeats, which have several conserved glycine and cysteine residues. Furthermore the A chain contains 20 internal cysteine-rich repeats present in tandem arrays in three separate clusters (domains IIIa, IIIb and V). Domain I + II has a predicted continuous alpha-helical structure characterized by heptad repeats and three domains (IVa, IVb and VI) are predicted to contain a number of beta-sheets and coiled-coil structures. Northern-blot analysis was used to study the laminin A chain expression in the JAR cell line, full-term placenta and newborn-human tissues (kidney, spleen, lung, heart muscle, psoas muscle and diaphragm muscle). The expression was detectable in newborn-human kidney and JAR cell line only. The overall amino acid sequence identity between human and mouse is 76%. The human chain has only one Arg-Gly-Asp (RGD) sequence, which is located in the long arm within domain G, whereas the single RGD sequence in the mouse chain is located in the short arm in domain IIIb. The degree of identity between the human laminin A chain sequence and the sequence available for merosin [Ehrig, Leivo, Argraves, Ruoslahti & Engvall (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3264-3268] is about 41% and when conservative substitutions are included the degree of similarity is 54%.

Amino Acid Sequence↗

PubMed1991

Constitutive expression of the mature array of neurofilament proteins by a CNS neuronal cell line.

Neurofilament protein expression was examined immunochemically in a neuronal cell line derived from postnatal day 21 septal tissue. The SN48.1p cell line was found to constitutively synthesize an array of neurofilament proteins typical of a mature neuron. All three neurofilament subunits (NF-L, NF-M, and NF-H) as well as differentially phosphorylated isoforms (P-, P+, P++, and P ) of NF-M and NF-H were identified by immunoblot analysis. Immunofluorescence studies revealed that the neurofilament proteins were components of discrete, filamentous structures. Abnormal intracellular aggregations of neurofilament proteins were never observed. Some SN48.1p cells apportioned specific isoforms into selected intracellular regions based on the molecular weight and phosphorylation level of the protein. NF-L was preferentially localized to perikarya and proximal neurites; NF-M[P++] and NF-H[P ] were distributed to distal aspects of neurites. The expression of these differentiated features of neurofilament proteins and, presumably, the synthesis of the kinases and phosphatases required for normal neurofilament metabolism occurred in the absence of growth factors, differentiating agents, and specialized culture substrates. In addition, the non-neuronal intermediate filaments glial fibrillary acidic protein and epithelial cytokeratin proteins were absent. These data demonstrate that SN48.1p cells exhibit a neurofilament phenotype characteristic of mature neurons and provide a unique model to examine the expression and function of neurofilaments in differentiated neuronal cells.

Animals↗

PubMed1985

The development of type I and type II benzodiazepine receptors in the mouse cortex and cerebellum.

The postnatal development of benzodiazepine (BDZ) receptors was monitored in Heterogeneous Stock (HS) mice, and the BDZ receptors were characterized and categorized into Type I and Type II receptors. When the number of 3H-Flu binding sites (Bmax) was assessed at weekly intervals after the birth of the animal, the number of sites in both the cortex and cerebellum increased significantly if the data was expressed as fmol/mg tissue. On the other hand, no significant change in 3H-Flu binding sites was evidenced in the cortex, and the number of 3H-Flu binding sites in the cerebellum decreased during postnatal development if Bmax values were expressed as fmole/mg protein. When receptor binding data was analyzed for the presence of Type I and Type II BDZ receptors, the changes in KD values for 3H-Flu binding development could be accounted for by changes in relative proportions of Type I and Type II receptors present in the cortex and cerebellum during the maturation process. Type II receptors predominated in both cortex and cerebellum at birth, and Type I receptors proliferated primarily during the first two weeks of postnatal life. In the cortex of adult mice there were approximately equal numbers of Type I and Type II BDZ receptors. In the cerebellum of adult mice, computer assisted analysis of binding data could not distinguish the presence of two distinct BDZ binding sites. However, Hill coefficients and overall binding constants determined from data on CL-218,872 displacement of 3H-Flu binding to cerebellar membranes indicated that cerebellar tissue from adult mice did contain a heterogeneous array of BDZ receptors.

Aging↗

PubMedDate not supplied

NF-I/Sp1 switch elements regulate collagen alpha 1(I) gene expression.

The expression of type I collagen is regulated developmentally and tissue specifically. Two sets of binding sites for nuclear factor I (NF-I) and Sp1 transcription factors arrayed as an imperfect tandem repeat are critical for high activity of the murine alpha 1(I) collagen gene in NIH-3T3 fibroblasts and are conserved in evolution. Gel retardation analysis combined with methylation interference studies show that NF-I and Sp1 bind to overlapping sites in a mutually exclusive manner. Cotransfection studies using Drosophila Schneider L2 cells, which lack both transcription factors, demonstrate that each factor alone trans-activates the gene, while cotransfection of both factors results in the inhibition of the strong Sp1 trans-activation. In contrast, the herpes simplex virus thymidine kinase promoter, which contains functionally independent NF-I and Sp1 binding sites, is maximally transactivated by the cotransfection of both factors. Because the two NF-I/Sp1 binding sites overlap, the ratio of the activities of the two factors rather than their absolute concentrations determine alpha 1(I) gene expression, characterizing these promoter sequences as transcription factor switch elements.

3T3 Cells↗