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Biomedical subjects

S M Cohen

Publications and source records attributed to S M Cohen.

At least 55 records · Page 3Linked to original sources
PubMed2000

Enhanced binding of the TATA-binding protein to TATA boxes containing flanking cisplatin 1,2-cross-links.

The TATA-binding protein (TBP) is essential for transcription initiation in eukaryotes. TBP recognizes and binds to the minor groove of a consensus sequence, TATAAA, known as the TATA box or TATA element. DNA binding is affected largely by hydrophobic contacts and through the intercalation of two sets of adjacent phenylalanine residues. The resultant duplex is sharply kinked, bending toward the major groove. Inspired by prior structural information showing intercalation of a phenylalanine side chain of a high mobility group (HMG) domain into the site of a cisplatin 1, 2-intrastrand d(GpG) cross-link, a series of DNA probes was prepared with one or two such adducts flanking the TATA box positions at or near the sites of TBP intercalation. The platinum adducts bend the DNA toward the major groove and result in as much as a 175-fold increase in binding affinity of the TBP over the unmodified target sequence. Kinetic studies indicate that the enhanced binding to the modified TATA box is predominantly a consequence of a >30-fold slower dissociation rate of the protein-platinated DNA complex. This work demonstrates that it is feasible to design rationally and to synthesize an enhanced affinity-binding site for a sequence-specific DNA-binding protein by appropriate chemical modification of flanking sequences. It also has implications for the mechanism of action of cisplatin.

Binding, Competitive↗
PubMed2000

Construction of a cosmid library of DNA replicated early in the S phase of normal human fibroblasts.

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by release from confluence arrest and incubation in the presence of aphidicolin. Bromodeoxyuridine (BrdUrd) was added to aphidicolin-containing medium to label DNA replicated as cells entered S phase. Nuclear DNA was partially digested with Sau 3AI, and hybrid density DNA was separated in CsCl gradients. The purified early-replicating DNA was cloned into sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High-density colony hybridization filters (1, 536 clones/filter) were prepared from a set of replicas of the original plates. Bacteria remaining in the wells of replica plates were combined, mixed with freezing medium, and stored at -80 degrees C. These pooled stocks were analyzed by polymerase chain reaction to determine the presence of specific sequences in the library. Hybridization of high-density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96-well plates. In testing the library for the presence of 14 known early-replicating genes, we found sequences at or near 5 of them: APRT, beta-actin, beta-tubulin, c-myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication.

Aphidicolin↗
PubMed2000

Wingless gradient formation in the Drosophila wing.

BACKGROUND: Secreted signaling proteins of the Wingless (Wg)/Wnt, Hedgehog and bone morphogenetic protein (BMP)/Decapentaplegic (Dpp) families function as morphogens to control growth and pattern formation during development. Although these proteins have been shown to act directly on distant cells in the developing limbs of the fruit fly Drosophila, little is known about how ligand gradients form in vivo. Wg protein is found in vesicles in Wg-responsive cells in the embryo and imaginal discs. It has been proposed that Wg may be transported by a vesicle-mediated mechanism. RESULTS: A novel method to visualize extracellular Wg protein was used to show that Wg forms an unstable gradient on the basolateral surface of the wing imaginal disc epithelium. Wg movement did not require internalization by dynamin-mediated endocytosis. Dynamin activity was, however, required for Wg secretion. By reversibly blocking Wg secretion, we found that Wg moves rapidly to form a long-range extracellular gradient. CONCLUSIONS: The Wg morphogen gradient forms by rapid movement of ligand through the extracellular space, and depends on continuous secretion and rapid turnover. Endocytosis is not required for Wg movement, but contributes to shaping the gradient by removing extracellular Wg. We propose that the extracellular Wg gradient forms by diffusion.

Animals↗
PubMed2000

Transdifferentiation of somatotrophs to thyrotrophs in the pituitary of patients with protracted primary hypothyroidism.

In patients with protracted primary hypothyroidism, the pituitary is enlarged due to the lack of feedback inhibition by thyroid hormone. In the present work, adenohypophysial biopsies from three women with protracted primary hypothyroidism were investigated by routine histology, immunocytochemistry, double immunostaining, immunoelectron microscopy, and combined immunocytochemistry - in situ hybridization. These methods confirmed the presence of massive thyrotroph hyperplasia and the formation of "thyroidectomy" or "thyroid deficiency" cells. A number of thyroidectomy cells were found to be immunoreactive for growth hormone (GH). Double immunostaining and immunoelectron microscopy revealed the presence of bihormonal cells containing both GH and thyroid stimulating hormone (TSH). Immunostaining combined with in situ hybridization revealed GH immunoreactive cells expressing TSH mRNA as well as TSH immunopositive cells expressing GH mRNA. Our findings provide conclusive evidence that somatotrophs may transform to thyrotrophs. Thus, in addition to multiplication of thyrotrophs, transdifferentiation of GH cells to thyrotrophs contributes to the increase of TSH-producing cells. The presence of such bihormonal cells best termed "thyrosomatotrophs" supports the concept that adenohypophysial cells are not irreversibly committed to the production of one single hormone and that their phenotype can change in response to functional demand.

Adult↗
PubMed2000

Proximal distal axis formation in the Drosophila leg: distinct functions of teashirt and homothorax in the proximal leg.

The proximal distal axis of the Drosophila leg is patterned by expression of a number of transcription factors in discrete domains along the axis. The homeodomain protein Homothorax and the zinc-finger protein Teashirt are broadly coexpressed in the presumptive body wall and proximal leg segments. Homothorax has been implicated in forming a boundary between proximal and distal segments of the leg. We present evidence that Teashirt is required for the formation of proximal leg segments, but has no role in boundary formation.

Animals↗
PubMed2000

Epidemiology and etiology of premalignant and malignant urothelial changes.

Bladder neoplasms are common around the world. Incidences are particularly high in the Nile River Valley secondary to schistosomiasis, which is frequently associated with the development of squamous cell carcinoma similar to that of other chronic inflammatory processes of the lower urinary tract. However, elsewhere, most bladder tumors are of the urothelial (transitional) cell type. There is a marked male predominance and there are extensive racial differences. It is predominantly a neoplasm that occurs in patients aged >50 years. Urothelial carcinomas comprise two distinct diseases both biologically and molecularly: a low-grade papillary tumor which frequently recurs; and a high-grade malignancy which can present as dysplasia or carcinoma in situ, but frequently presents as invasive disease. However, epidemiological investigations of urothelial malignancies have generally not distinguished between preneoplastic and invasive neoplasms or between these two types of urothelial neoplasms. It is recommended that future studies should distinguish between these entities. The most common etiologic factor of urothelial malignancies besides schistosomiasis is cigarette smoking. In addition, numerous specific chemicals have been identified as bladder carcinogens in humans, some relating to specific occupational exposures. Bladder carcinogens include aromatic amines and amides, such as 4-aminobiphenyl, benzidine, 2-naphthylamine and phenacetin-containing analgesics, and certain cancer chemotherapeutic agents, such as phosphoramide mustards. More recently, occupational exposure to various combustion gases, such as diesel exhaust, has been related to an increased risk of developing bladder neoplasms. Also, exposure to chlorination by-products in drinking water and to arsenic has been suggested as increasing the risk of bladder neoplasia. As numerous specific chemicals appear to be related to the development of bladder tumors, various polymorphisms of enzymes involved in their metabolism have been suggested as affecting the susceptibility to their carcinogenicity. This has been particularly true with respect to the role of acetyltransferases in relation to aromatic amine carcinogenesis. Dietary influences have also been suggested as affecting bladder neoplasia susceptibility. Various heterocyclic amines generated by pyrolysis of food have been suggested as potential dietary factors increasing the risk of bladder cancer, particularly in relation to the ingestion of red meat. Despite the existence of several identifiable factors that increase or decrease the risk of bladder cancer, many patients have no known carcinogens or risk factors.

Adenocarcinoma↗
PubMed2000

Prevention and treatment of urothelial premalignant and malignant lesions.

Bladder cancer is believed to develop through reversible premalignant stages followed by irreversible steps, and ending in invasive cancer giving rise to distant metastases. Because of the variation in the clinical course it has also been suggested that different forms of cancer develop along different molecular pathways leading to tumor presentations of various malignant potential. Today we treat and prognosticate bladder cancer on the basis of clinical and histologic findings that are insufficient to assess all the biologic potential of these tumors. Understanding the pathogenesis of bladder cancer might lead to a more precise identification of particular tumors with regard to clinical aggressiveness, resulting in individualized strategies for treatment and prophylaxis. Bladder cancer is seldom diagnosed in its preclinical stage, it is instead detected at cystoscopy and virtually never recognized as an incidental finding on autopsy. Therefore its "natural history" largely reflects that of "treated" disease. The true incidence of premalignant and malignant epithelial changes is not known. Incidences of hyperplasia and dysplasia of approximately 10% and approximately 5%, respectively and only occasional findings of cancer itself were reported in two autopsy series. Urothelial dysplasia is generally believed to be premalignant and a putative precursor of invasive cancer but unfortunately there has been a lack of standardization in terms of terminology and diagnosis. There is also a need for an agreed definition of the boundary between premalignancy, i.e. urothelial changes that have some but not all the features of carcinoma in situ, and malignancy, especially when considering potentially harmful treatments to prevent this transition. Most new diagnostic tools available and being tested today compare new detection techniques with traditional methods such as cytology or conventional histology of malignant rather than premalignant changes. There is probably also a short preclinical latency, as implied by the incidental findings of bladder cancer at autopsy, which makes it necessary to define how and when to promote early detection and treatment. Future studies therefore have to concentrate on methods for early detection of disease as well as characterization of host susceptibility, evaluation of exposure to carcinogens and potential effects of preventive measures. It is also likely that the improved tools of molecular prognostication will allow us to design trials more precisely in order to tailor therapeutic strategies.

Animals↗
PubMed2000

Calcium phosphate-containing precipitate and the carcinogenicity of sodium salts in rats.

Sodium saccharin, ascorbate and other sodium salts fed at high doses to rats produce urinary bladder urothelial cytotoxicity with consequent regenerative hyperplasia. For sodium salts that have been tested, tumor activity is enhanced when administered either alone or after a brief exposure to a known genotoxic bladder carcinogen. These sodium salts alter urinary composition of rats resulting in formation of an amorphous precipitate. We examined the precipitate to ascertain its composition and further delineate the basis for its formation in rat urine. Using scanning electron microscopy with attached X-ray energy dispersive spectroscopy, the principal elements present were calcium, phosphorus, minor amounts of silicon and sulfur. Smaller elements are not detectable by this method. Infrared analyses demonstrated that calcium phosphate was in the tribasic form and silicon was most likely in the form of silica. Small amounts of saccharin were present in the precipitate from rats fed sodium saccharin (<5%), but ascorbate was not detectable in the precipitate from rats fed similar doses of sodium ascorbate. Large amounts of urea and mucopolysaccharide, apparently chondroitin sulfate, were detected in the precipitate by infrared analysis. Chemical analyses confirmed the presence of large amounts of calcium phosphate with variably small amounts of magnesium, possibly present as magnesium ammonium phosphate crystals, present in urine even in controls. Small amounts of protein, including albumin and alpha(2u)-globulin, were also detected (<5% of the precipitate). Calcium phosphate is an essential ingredient of the medium for tissue culture of epithelial cells, but when present at high concentrations (>5 mM) it precipitates and becomes cytotoxic. The nature of the precipitate reflects the unique composition of rat urine and helps to explain the basis for the species specificity of the cytotoxic and proliferative effects of high doses of these sodium salts.

Animals↗
PubMed2000

Lessons learned in applying the U.S. EPA proposed cancer guidelines to specific compounds.

An expert panel was convened to evaluate the U.S. Environmental Protection Agency's "Proposed Guidelines for Carcinogen Risk Assessment" through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel's activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical's mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted.

Animals↗
PubMed2000

Reevaluation of the cancer potency factor of toxaphene: recommendations from a peer review panel.

This reevaluation of the current U.S. EPA cancer potency factor for toxaphene is based upon a review of toxaphene carcinogenesis bioassays in mice conducted by Litton Bionetics (unpublished report, 1978) and the National Cancer Institute (NCI) (Technical Report Series No. 37, conducted by Gulf South Research Institute, 1979). The mechanistic data available for toxaphene, including consideration of the potential of the compound to induce genotoxicity, was examined with an emphasis on whether this information supports a change in the cancer potency factor. If a quantitative dose-response assessment for toxaphene is to be performed, the data from both the NCI and Litton cancer bioassays should be used. Additionally, liver tumor results from female mice, rather than male mice, should be used for estimating potential human cancer risk because the background rate of liver tumors in females is lower and less variable than that exhibited by males. An ED(10) was estimated as the point of departure. The mechanistic data were not sufficient to fully support a margin of exposure approach. Therefore, we believe that applying a linear extrapolation from the ED(10) to the origin is an appropriate means to estimate risk at low doses. This is a highly conservative approach and, when it is applied, we conclude that the current EPA cancer potency factor should be reduced from 1.1 (mg/kg/day)(-1) to 0.1 (mg/kg/day)(-1).

Animals↗
PubMed2000

Therapeutic use of selected herbs.

An increasing number of people in the United States are using herbs for health promotion and specific symptom management. Herbs are used to initiate healing through synergistic responses unlike the specific properties of pharmaceuticals. Anecdotal data comprise much of the popular information available about herbs. Scientific studies of the efficacy and safety of herbs, although on the rise, are less available than other drug trials. Clinicians need an appropriate knowledge base for dealing with patients who take herbal preparations as well as the ability to confidently include herbal preparations in their formulary. In this article, five common herbs are reviewed. The effects, clinical studies, side effects, and dosing regimens for aloe vera, arnica, black cohosh, evening primrose oil, and saw palmetto are described.

Aloe↗
PubMed2000

Subcellular localization of leptin in non-tumorous and adenomatous human pituitaries: an immuno-ultrastructural study.

Leptin is a key mediator in the maintenance of neuroendocrine homeostasis. Recently, leptin and leptin receptor expression were demonstrated in non-tumorous and adenomatous human pituitaries. This study was performed to determine the subcellular localization of leptin in human adenohypophyses (n = 3) and in various types of pituitary adenoma (n = 16). Immunoelectron microscopy showed leptin immunolabeling in most hormone-producing cells of the human non-tumorous adenohypophysis, but not in stellate cells. Labeling was noted over secretory granules. Immunocytochemistry using double labeling revealed leptin expression in GH-, ACTH-, TSH-, and FSH/LH-containing cells but not in PRL cells. The percentage of immunopositive cells and the intensity of immunostaining varied considerably among the various cell types. Immunoelectron microscopy with double gold labeling showed co-localization of leptin and adenohypophysial hormones in the same secretory granules. Among pituitary tumors, leptin immunolabeling was evident only in corticotroph adenomas. Compared to non-tumorous corticotrophs, leptin immunoexpression was less abundant in corticotroph adenomas. The presence of leptin and adenohypophysial hormones in the same secretory granules suggests that leptin is secreted concomitantly with various adenohypophysial hormones and that its release is under the control of hypothalamic stimulating and inhibiting hormones.

Adenoma↗
PubMed2000

Temporal regulation of apterous activity during development of the Drosophila wing.

Dorsoventral axis formation in the Drosophila wing depends on the activity of the selector gene apterous. Although selector genes are usually thought of as binary developmental switches, we find that Apterous activity is negatively regulated during wing development by its target gene dLMO. Apterous-dependent expression of Serrate and fringe in dorsal cells leads to the restricted activation of Notch along the dorsoventral compartment boundary. We present evidence that the ability of cells to participate in this Apterous-dependent cell-interaction is under spatial and temporal control. Apterous-dependent expression of dLMO causes downregulation of Serrate and fringe and allows expression of delta in dorsal cells. This limits the time window during which dorsoventral cell interactions can lead to localized activation of Notch and induction of the dorsoventral organizer. Overactivation of Apterous in the absence of dLMO leads to overexpression of Serrate, reduced expression of delta and concomitant defects in differentiation and cell survival in the wing primordium. Thus, downregulation of Apterous activity is needed to allow normal wing development.

Animals↗
PubMed2000

On the relationship of matrix association and DNA replication.

The nuclear matrix is believed to contain sites of assembly of protein complexes that catalyze the initiation of DNA replication as well as DNA elongation. To explore this relationship, DNA replicated by human fibroblasts at the beginning of the S phase was purified and used to construct a cosmid library. Hybridization studies with a subgroup of clones (about one-sixth of the total clones in this library) showed that many of them were highlighted by probes prepared from early replicating DNA, as well as from nuclear matrix-associated DNA. Statistical analysis showed a positive correlation between these hybridization results. We seek to identify origins of replication that are activated early in the S phase of the cell cycle in human cells. Therefore, clones isolated from this library are being analyzed for the presence of structural motifs that have been found in other origins of replication and for potential sites of attachment to the nuclear matrix. This method of analysis is illustrated here using the published sequences for the origins of replication reported for the human lamin B2 and HPRT genes.

DNA Replication↗
PubMed1999

New growth factors for imaginal discs.

Several families of peptide growth factors are implicated in regulating cell growth and proliferation of vertebrate cells in culture. Genetic studies in Drosophila implicate some of these factors in growth control in vivo. A recent report identifies a new family of growth factors, related to chitinase enzymes, required by Drosophila imaginal disc cells in culture. It will be of interest to determine how such factors relate to size regulation during development.

Animals↗
PubMed1999

Formation of morphogen gradients in the Drosophila wing.

Patterning of multicellular fields requires mechanisms to coordinate developmental decisions made by populations of cells. Evidence is accumulating that the necessary information is provided by localized sources of secreted signalling proteins which act as morphogens. We review evidence that Wingless, Dpp and Hedgehog proteins act as morphogens in the developing wing of Drosophila and discuss recent work illustrating that signalling helps to shape their activity gradients by regulating ligand distribution and by modulating the responsiveness of target cells. These studies suggest that there is more to being a morphogen than formation of a ligand gradient by passive diffusion.

Animals↗