Biomedical subjects

M M Sanders

Publications and source records attributed to M M Sanders.

At least 37 records · Page 2Linked to original sources

PubMed1996

Identification of topoisomerase I as the cytotoxic target of the protoberberine alkaloid coralyne.

Protoberberine alkaloids (coralyne and its derivatives), which exhibit antileukemic activity in animal models, have been shown to be potent inducers of topoisomerase (topo) I-DNA cleavable complexes using purified recombinant human DNA topo I. Different from the structurally similar benzophenanthridine alkaloid nitidine (a dual poison of both topos I and II), coralyne and its derivatives have marginal poisoning activity against DNA topo II. Yeast cells expressing human DNA topo I are shown to be specifically sensitive to killing by coralyne derivatives and nitidine, suggesting that cellular DNA topo I is their cytotoxic target. Two human camptothecin-resistant cell lines, CPT-K5 and A2780/CPT-2000, which are known to express highly camptothecin-resistant topo I, are only marginally resistant to coralyne derivatives and nitidine. Purification of human topo I from Escherichia coli cells overexpressing CPT-K5 recombinant topo I has demonstrated similar marginal cross-resistance to nitidine. It seems possible to develop coralyne and nitidine derivatives as new topo I-targeted therapeutics to overcome aspects of camptothecin-related resistance.

Animals↗

PubMed1996

Regulation of the chicken ovalbumin gene by estrogen and corticosterone requires a novel DNA element that binds a labile protein, Chirp-1.

Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE.

Animals↗

PubMed1996

Ten years after: reclassification of steroid-responsive genes.

Although several hundred genes are directly or indirectly regulated by steroid hormones, significant gaps exist in our understanding of the relevant mechanisms, particularly for those genes that do not directly bind intracellular receptors or that exhibit delayed changes in transcription rates upon receptor binding. To assist in defining the mechanism of action of steroid hormones, we are proposing that a standard nomenclature be adopted for classifying steroid-responsive genes, based upon whether the receptors directly bind to the target genes and the kinetics of the response. Three categories are proposed: primary response genes, delayed primary response genes, and secondary response genes.

Animals↗

PubMed1995

Repression of the ovalbumin gene involves multiple negative elements including a ubiquitous transcriptional silencer.

Most eukaryotic genes are controlled by a complex array of cis-acting regulatory elements that modulate transcriptional activity. Two major regulatory elements reside in the chicken ovalbumin gene, a steroid-dependent regulatory element (SDRE, -892 to -780) and a negative regulatory element (NRE, -308 to -88). The SDRE is required for responsiveness to estrogen and glucocorticoid. The NRE appears to have the dual role of repressing transcription in the absence of steroids and of cooperating with the SDRE to activate transcription in the presence of steroids. The experiments described herein were designed to investigate the role of the NRE in repressing gene expression. Transfection of OvCAT fusion genes containing deletions in the NRE into primary oviduct cell cultures identified three elements (-308 to -256, -239 to -220, and -174 to -88) that repress transcription. Oligomers corresponding to portions of these elements also independently repress the viral thymidine kinase promoter. Interestingly, the element from -239 to -220 functions mechanistically as a silencer and shares sequence identity with silencers in other genes (TCTCTCCNA). Mobility shift studies indicated that all of the negative elements bind specific protein complexes from oviduct, none of which is appreciably affected by treatment with steroid hormones. However, oviduct-specific proteins bind to the regions from -280 to -252 and from -134 to -88, providing the first identification of potential tissue-specific elements in the ovalbumin gene. These results demonstrate that the region of DNA originally called the NRE is a multifunctional regulatory element that may be involved in several diverse regulatory activities.

Animals↗

PubMed1993

A complex array of double-stranded and single-stranded DNA-binding proteins mediates induction of the ovalbumin gene by steroid hormones.

The transcriptional induction of the chicken ovalbumin gene by steroid hormones is abolished by inhibitors of protein synthesis such as cycloheximide, suggesting that a labile protein mediates this process. A steroid-dependent regulatory element (SDRE) has been identified in the 5'-flanking region of the gene between -900 and -780 that is required for induction by steroids. Additional transfection experiments limit the 5'-border of the SDRE to the region between -892 and -864. To investigate whether any of the proteins binding to the SDRE are affected by estrogen or cycloheximide, protein binding was investigated using DNase I and exonuclease III footprinting and gel mobility shift assays. These experiments demonstrate that labile proteins bind to the sequences between -900 and -860 and between -810 and -820. Four oviduct nuclear proteins, including one of the labile proteins, binding to the SDRE prefer single-stranded DNA (ssDNA) in a sequence-specific manner. The binding activity of three of these ssDNA-binding proteins is increased in oviduct nuclear protein extracts from estrogen-treated chicks. These data suggest that induction of the ovalbumin gene is mediated by a complex collection of ssDNA- and double-stranded DNA-binding proteins whose activities are in turn regulated by their short half-lives or by estrogen.

Animals↗

PubMed1993

A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein.

A novel intermediate filament cDNA, pG-IF, has been isolated from a Drosophila melanogaster embryonic expression library screened with a polyclonal antiserum produced against a 46 kDa cytoskeletal protein isolated from Kc cells. This 46 kDa protein is known to be immunologically related to vertebrate intermediate filament proteins. The screen resulted in the isolation of four different cDNA groups. Of these, one has been identified as the previously characterized Drosophila nuclear lamin cDNA, Dm0, and a second, pG-IF, demonstrates homology to Dm0 by cross hybridization on Southern blots. DNA sequence analysis reveals that pG-IF encodes a newly identified intermediate filament protein in Drosophila. Its nucleotide sequence is highly homologous to nuclear lamins with lower homology to cytoplasmic intermediate filament proteins. pG-IF predicts a protein of 621 amino acids with a predicted molecular mass of 69,855 daltons. In vitro transcription and translation of pG-IF yielded a protein with a SDS-PAGE estimated molecular weight of approximately 70 kDa. It contains sequence principles characteristic of class V intermediate filament proteins. Its near neutral pI (6.83) and the lack of a terminal CaaX motif suggests that it may represent a lamin C subtype in Drosophila. In situ hybridization to polytene chromosomes detects one band of hybridization on the right arm of chromosome 2 at or near 51A. This in conjunction with Southern blot analysis of various genomic digests suggests one or more closely placed genes while Northern blot analysis detects two messages in Kc cells.

Amino Acid Sequence↗

PubMed1993

Influence of the estrous cycle on the development of upper genital tract pathology as a result of chlamydial infection in the guinea pig model of pelvic inflammatory disease.

Guinea pigs were infected intravaginally with the chlamydial agent of guinea pig inclusion conjunctivitis at varying times during the estrous cycle. Genital tract tissues were collected 30 days after infection and processed for histopathological analysis. No difference was seen in the course of lower genital tract infection. However, a significantly greater percentage of tissues from animals infected on day 11 of the cycle were found to have chronic inflammation and fibrosis in the mesosalpinx compared to those from animals infected on day 6 or day 16. In addition, a significantly greater percentage of oviduct tissues from day 11-infected guinea pigs had marked tubal dilatation when compared to oviducts from day 6-or day 16-infected animals. The increased incidence of pathological changes was also noted in the endocervix, uterine fundus, and uterine horns but not the exocervix. These data indicate that the time of the estrous cycle and the corresponding hormonal influences may be an important influence on the development of upper genital tract disease.

Animals↗

PubMed1992

Glutamine and glutamate metabolism in normal and heat shock conditions in Drosophila Kc cells: conditions supporting glutamine synthesis maximize heat shock polypeptide expression.

We have previously reported that Drosophila Kc cells require glutamine for maximal expression of heat shock proteins in stressed conditions (Sanders and Kon: J. Cell. Physiol. 146:180-190, 1991). The mechanism of this effect has been investigated by comparing the metabolic utilization of glutamine in conditions which support hsp expression with that of glutamate in conditions where up to 100-fold less hsp is synthesized. This comparison showed that free ammonia was generated by cells incubated in the presence of glutamine in 37 degrees C (heat shock) conditions, but not at 25 degrees C, and not in the presence of glutamate in either normal or heat shock conditions. There was no difference in the amount of [14C]O2 generated from either [14C]-labeled amino acid in the tricarboxylic acid cycle, but three- to four-fold more alanine was synthesized in cells incubated in glutamine than in glutamate. Treating the cells with aminotransferase inhibitors to artificially increase NH3 release raised hsp expression in the presence of glutamate to maximal levels characteristic of glutamine. This potentiation correlated with inhibition of alanine aminotransferase. Since only NH3 production correlated with hsp expression in heat shock conditions in the presence of glutamine, and NH3 addition to glutamate also resulted in maximal hsp expression, we measured glutamine production in glutamate plus NH3 and observed net glutamine synthesis. The supposition that glutamine itself is responsible for the regulatory changes supporting maximal hsp expression was supported by the finding that the glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), mimicked the effects of glutamine. We conclude that glutamine imposes regulatory changes which alter nitrogen metabolism and support hsp expression in Kc cells.

Amino Acids↗

PubMed1992

Regulation of expression of the chicken ovalbumin gene: interactions between steroid hormones and second messenger systems.

The chicken ovalbumin gene is subject to multihormonal regulation. Maximal expression of it requires not only the synergistic effects of estrogen and corticosterone, but also the permissive effects of insulin. In addition to effects on transcription, the stability of its message is greatly enhanced by estrogen. Furthermore, two signal transduction pathways involving protein kinases have been implicated in the regulation of the ovalbumin gene. To better define the role of second messengers on expression of the ovalbumin gene, the effects of the protein kinase-C (PKC) and the cAMP-dependent protein kinase (PKA) pathways on the endogenous levels of ovalbumin mRNA and the transcription of an ovalbumin fusion gene were investigated. Primary cultures of oviduct cells were treated with phorbol 12-myristilate 13-acetate (an activator of PKC) or with forskolin and 3-isobutyl-1-methylxanthine (an activator of PKA) alone, activators plus estrogen and corticosterone, or activators plus both steroids and insulin. The results indicate that phorbol 12-myristilate 13-acetate causes a dramatic destabilization of ovalbumin message, resulting in a reduction in ovalbumin mRNA levels. In contrast, the activators of the PKA system can substitute for insulin and, thereby, increase expression of the ovalbumin gene synergistically with the steroids. The effect of the activators of the PKA system is at the level of transcription. Thus, in chicken oviduct cell cultures, the PKA and PKC signal transduction pathways act in opposing ways to modulate the steroid-induced expression of the ovalbumin gene.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Pathogenesis of endometritis and salpingitis in a guinea pig model of chlamydial genital infection.

The development of tubal obstruction and subsequent infertility is a major sequelum of upper genital tract infection with Chlamydia trachomatis; however, little is known about the pathogenesis of the infection. In this investigation, the authors present a detailed study of the progression of ascending chlamydial infection in female guinea pigs resulting from intravaginal inoculation of the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC). Isolation of chlamydiae from different tissues of the genital tract revealed definitive evidence for ascending infection that was not dose-related. By 7 days after infection, GPIC was isolated from the endometrium and oviducts of 78% of the animals. Pathologic changes analogous to those seen in human chlamydial disease, including polymorphonuclear, mononuclear, and plasma cell infiltration, were seen in the endometrium and oviducts, although not all isolation positive animals developed overt tubal disease. Long-term fibrosis, often in combination with hydrosalpinx, was noted in the mesosalpingeal tissue in 20% of the animals. Thus, the guinea pig:GPIC system represents a model for ascending chlamydial infection resulting from vaginal inoculation of normal guinea pigs that closely approximates the disease as seen in humans and can be used to study the pathogenesis of chlamydial genital infection.

Animals↗

PubMed1991

A protein with a binding specificity similar to NF-kappa B binds to a steroid-dependent regulatory element in the ovalbumin gene.

The chicken ovalbumin gene is regulated at the level of transcription by four classes of steroid hormones. A steroid-dependent regulatory element (SDRE) found from -900 to -732 is required for this steroid-mediated induction. To define more precisely sequences of the SDRE required for steroidal induction, a series of exonuclease III deletions were made in the 3' end of the SDRE. Fusion genes containing the mutant ovalbumin 5'-flanking sequences linked to the chloramphenicol acetyltransferase structural gene (CAT) were transfected into steroid-responsive primary oviduct cells. These functional studies defined a region of the SDRE from -793 to -759 that is essential for induction by steroids. Analysis of protein interactions in this 34-base pair region by copper-phenanthroline footprinting and methylation interference assays defined nucleotides required for protein binding. Footprinting showed protection of residues extending from -784 to -765, an area that included nucleotides that, when methylated, interfered with protein binding. In addition, this footprinted region contained 10 nucleotides that were identical to sequences contained in the beta-interferon gene regulatory element. An oligomer synthesized to this region of homology produced two DNA-protein complexes with oviduct nuclear proteins. Although this region of the interferon gene regulatory element binds the transcription factor NF-kappa B, an oligomer from the immunoglobulin kappa light chain gene known to bind NF-kappa B did not compete with the SDRE oligomer for binding to oviduct nuclear proteins. Surprisingly, this same NF-kappa B oligomer was able to restore steroid responsiveness to an SDRE mutant, while an oligomer from the immunoglobulin heavy chain gene inserted in the same position did not affect induction by steroids. These data suggest that a protein binding to sequences in the SDRE that are similar to an NF-kappa B-binding site participates in the steroid-mediated increase in transcription of the chicken ovalbumin gene.

Animals↗

PubMed1991

Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.

Amino Acids↗

PubMed1990

The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element.

Transcription of the chicken ovalbumin gene is induced both in vivo and in vitro by four classes of steroid hormones. Recent experiments identified a steroid-dependent regulatory element (SDRE) in the 5'-flanking region of the ovalbumin gene between -900 and -521. To characterize the regulatory properties of the SDRE more precisely, additional mutations were created in this region, and fusion genes prepared by linking the ovalbumin 5'-flanking region and promoter to the chloramphenicol acetyltransferase structural gene. When the ovalbumin-chloramphenicol acetyltransferase fusion genes were transfected into steroid-responsive primary oviduct cells, mutants lacking sequences between -900 and -732 were no longer responsive to estrogen, corticosterone, progesterone, or dihydrotestosterone. The SDRE did not confer steroid-dependent expression on the heterologous thymidine kinase promoter by itself but did in conjunction with the negative regulatory element identified between -350 and -100. This suggests that the two elements act as a single functional entity and that the SDRE is not behaving as a typical steroid response element. Gel shift analyses revealed that two SDRE.protein complexes were formed when nuclear protein extracts were derived from estrogen-treated chicken oviduct but that only one complex was formed with extracts from estrogen-withdrawn oviduct or from other tissues. Neither an estrogen response element oligomer nor a glucocorticoid/progesterone response element oligomer competed for either of the DNA.protein complexes. Partially purified progesterone receptor also did not bind to the SDRE. These data indicate that induction of the ovalbumin gene by steroid hormones requires complex interactions involving both the SDRE and the negative regulatory element.

Animals↗

PubMed1990

Distribution of CA 125 in embryonic tissues and adult derivatives of the fetal periderm.

New murine monoclonal antibodies to a partially purified CA 125 antigen were developed and identified as M 2 and M 11. With immunohistochemical techniques, these new antibodies and OC 125 antibody were used to search for CA 125 in embryonic tissues and adult apocrine sweat glands and mammary glands. The embryonic skin, the periderm, expressed CA 125 antigen and its adult derivatives, the mammary glands and apocrine sweat glands, expressed CA 125 while in the active state of secretion. In a 6-week-old formalin-fixed and paraffin-embedded ectopic embryo specimen, antibodies M 2 and M 11 recognized CA 125 in the periderm, the notochord, the myocardium, the pericardium, the gastroenteric tract, enteric duct remnants in the umbilical cord (vitelline and allantoic ducts), the mesonephric duct, and the amnion. OC 125 staining of these formalin-fixed specimens was either very faint or absent. In a formalin-fixed and paraffin-embedded specimen of apocrine sweat glands from the axilla, antibodies M 2 and M 11 detected CA 125 antigen intracellularly in the secretory cells. Again no staining was observed with OC 125 antibody. In a frozen and acetone-fixed specimen of lactating mammary glands, antibodies M 2 and OC 125 detected CA 125 antigen intraductally. Colostrum and milk collected from 25 mothers at various stages post partum had mean CA 125 levels of 34,213 U/ml in colostrum, 1469 U/ml at 3 to 7 days, and 105 U/ml at 5 to 26 weeks.

Amnion↗

PubMed1989

Thyroid hormone and circadian regulation of the binding activity of a liver-specific protein associated with the 5'-flanking region of the S14 gene.

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.

Animals↗

PubMed1989

Overexpression of a 123-kDa anion transport inhibitor binding protein and two cytoskeleton proteins in Drosophila Kc cell variants resistant to disulfonic stilbenes.

Drugs of the disulfonic stilbene class, which inhibit anion transport in the cell membrane in many cell types, have been found to inhibit anion transport and cell growth in Drosophila Kc cells. Cell variants selected by a stepwise selection protocol for the ability to grow in the presence of the disulfonic stilbenes are severalfold resistant to growth inhibition by the drugs. Both the resistant populations and a cloned cell line show dramatic overexpression of three polypeptides. The most highly overproduced protein is a 123-kDa plasma membrane protein which binds the reversible anion transport inhibitor, flufenamic acid, in a protection biotinylation experiment. The 123-kDa putative anion transport protein copurifies with, and immunologically cross-reacts with, two detergent-insoluble cytoskeleton proteins of 46- and 62-kDa molecular weight, which are each overexpressed more than 8-fold in the variants. Resistance to growth inhibition by the disulfonic stilbenes and amplified expression of the 123-, 62-, and 46-kDa proteins are simultaneously lost over a period of 30 weeks in the absence of selective conditions, suggesting that the function of the overproduced polypeptides is related to growth control in Drosophila cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1989

Role of cell-mediated immunity in the resolution of secondary chlamydial genital infection in guinea pigs infected with the agent of guinea pig inclusion conjunctivitis.

Guinea pigs which have recovered from a genital infection with the agent of guinea pig inclusion conjunctivitis demonstrate strong immunity to reinfection for a short period of time but then become susceptible to reinfection. The secondary infection is markedly shortened in duration and decreased in intensity. Previous studies have indicated an important role for humoral immunity in resistance to and in recovery from reinfection. However, the contribution of cell-mediated immunity to immunity toward or recovery from a secondary infection is not clear. Guinea pigs were infected in the genital tract with guinea pig inclusion conjunctivitis and were challenged at either 30 or 75 days after the primary infection. Prior to challenge, one group of animals were injected with rabbit anti-guinea pig thymocyte serum (ATS) while control groups received either normal rabbit serum or no treatment. Treatment was continued daily for the course of the experiment. On day 30, ATS-treated guinea pigs had a slightly higher rate of reinfection, and generally the infection persisted longer than in controls. On day 75, all animals became reinfected upon challenge, but control animals resolved their infections in 3 to 9 days. In contrast, most ATS-treated animals remained infected throughout the course of the experiment. Although the animals became reinfected, the levels of chlamydiae were much lower than those observed during the primary infection. ATS treatment abrogated T-cell responses, but serum and secretory antibody responses remained normal. Histopathological examination revealed some decrease in mononuclear infiltration of endocervical and uterine tissues in ATS-treated animals. These data indicate that previously infected guinea pigs require both cell-mediated immunity and humoral immunity for resolution of a challenge infection.

Animals↗

PubMed1988

Positive and negative regulatory elements control the steroid-responsive ovalbumin promoter.

Steroid hormones regulate the transcriptional activity of the chicken ovalbumin gene both in vivo and in cell culture. To identify the regulatory elements involved, primary oviduct cell cultures were transfected with constructs containing the promoter and 5'-flanking region of the ovalbumin gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. Induction of the OvCAT genes by estrogen, progesterone, or corticosterone mimics that of the endogenous ovalbumin gene, indicating that the transfected DNA is accurately regulated. Deletion analysis revealed that a steroid response element (SRE) resides between nucleotide coordinates -880 and -585 and that a negative regulatory element (NRE) resides between -350 and -248 in the ovalbumin gene. Thus, an NRE represses expression of the ovalbumin gene unless steroid hormones relieve this negative control through interactions involving a more distal SRE. Neither the SRE nor the NRE alone regulates the heterologous thymidine kinase promoter, suggesting either that they function as a single entity or that they are conditional regulatory elements. The NRE is functional in MCF-7 cells, but the SRE cannot be activated by steroids in this heterologous estrogen-responsive cell line. These data indicate that the steroid-receptor complex induces the ovalbumin gene through direct or indirect actions at an SRE to relieve represssion at an NRE.

Animals↗