Biomedical subjects
S Strickland
Publications and source records attributed to S Strickland.
Transient translational silencing by reversible mRNA deadenylation.
Tissue-type plasminogen activator (tPA) mRNA is stored, stable and untranslated, in the cytoplasm of fully grown primary mouse oocytes. Dormancy is associated with an unusually short poly(A) tail, and poly(A) tail elongation controls tPA mRNA translational activation during meiotic maturation. Here we show that the nuclear transcript of this mRNA is extensively polyadenylated and that primary oocytes contain a deadenylating activity capable of silencing the cytoplasmic message. The sequence determinants that control deadenylation and polyadenylation overlap; this AU-rich region thus serves as an adenylation control element (ACE). The translation of a reporter mRNA in primary oocytes is prevented upon inclusion of an ACE in its 3' untranslated region. Therefore, the stage-specific regulation of poly(A) tail length accounts for the regulated synthesis of tPA in oocytes, and reversible deadenylation provides a mechanism for the translational control of dormant mRNAs.
Heparin-like activity in porcine follicular fluid and rat granulosa cells.
Heparin-like activity is present in rat and porcine follicular fluids, as determined by measuring the acceleration of the inactivation of purified human thrombin by antithrombin III. The heparin-like activity is dose-dependent and specific to follicular fluid proteoglycans. Cartilage proteoglycans do not exhibit this activity at any of the concentrations tested. The activity of these macromolecules resides in the polysaccharide unit. Destruction of the protein core of the follicular fluid proteoglycans by alkaline borohydride treatment does not interfere with the "heparin-like" effect, whereas it is completely destroyed by digestion with purified heparinase. Incubation with chondroitinases has no effect. Granulosa cells which are the source of follicular fluid proteoglycans express biologically active heparin-like mucopolysaccharides. These molecules are produced under gonadotropin regulation and are associated with the cell surface material.
Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation.
The cytoplasmic polyadenylation element (CPE) is an AU-rich sequence in the 3'-untranslated region of many stored maternal mRNAs. The CPE directs the meiotic maturation-specific cytoplasmic polyadenylation and translational activation of these dormant mRNAs in Xenopus. The work presented here demonstrates that the CPE controls a similar regulation in mouse oocytes and utilizes the information to isolate novel maternal mRNAs by polymerase chain reaction (PCR). A degenerate CPE primer was used in an anchored PCR reaction with cDNAs from primary mouse oocytes. Clones were identified that contained the canonical polyadenylation signal AATAAA. A novel PCR test was then used to determine the polyadenylation state of the respective mRNAs before and after meiotic maturation. Two mRNAs, OM-1 and OM-2, are cytoplasmically polyadenylated upon maturation. Another mRNA is not polyadenylated during maturation, although it contains multiple CPE-like elements, indicating that this sequence element is not sufficient for adenylation during this time. Microinjection into primary oocytes of antisense oligodeoxynucleotides directed against OM-1 destroys the mRNA but does not appear to interfere with maturation in vitro. These experiments identify two novel maternal mRNAs and establish a simple strategy for isolating other maternal messages that control meiotic maturation, fertilization, and early mouse development.
The result of an educational intervention for physicians providing HIV-antibody testing and counseling.
Based upon our findings related to physicians' human immunodeficiency virus (HIV) antibody-testing practices in a multispecialty group practice, we randomly selected a group of family practice physicians and over six months provided them with patient education resources and information about HIV-antibody-testing guidelines and their own patient counseling practices. Following this intervention, a chart audit showed that documentation of patient counseling had doubled. We also noted positive, albeit minor, changes in obtaining informed consent. Physicians ordered fewer tests overall; however, we found a slight increase in testing for "inappropriate" reasons.
In vitro analysis of the tissue plasminogen activator promoter reveals a GC box-binding activity present in murine brain but undetectable in kidney and liver.
Tissue plasminogen activator (t-PA) mRNA levels are high in murine brain, lower in kidney, and undetectable in liver. Differences in t-PA mRNA levels are regulated in part at the transcriptional level. Brain, kidney, and liver nuclear extracts direct regulated transcription from the murine t-PA promoter in a manner that reflects the relative levels of t-PA gene expression in these tissues in vivo. Analysis of mutants has defined two GC box motifs as important elements for regulated transcription in vitro. Upon investigation of protein-DNA binding, we detected an activity in brain extracts which was not detected in kidney or liver extracts. An Sp1-like factor also binds to this region in all three tissue types. DNA interference experiments show that the brain-enriched binding activity and the Sp1-like factor contact the same GC-rich sequences. These studies provide additional evidence that brain-enriched DNA-binding activities can interact with sequences also recognized by ubiquitous transcription factors.
Occupational health nurses' attitudes toward cancer.
Attitudes toward cancer held by a study population of 100 occupational health nurses were not significantly different than a normative reference group and did not indicate a negative attitude toward cancer. The majority of the literature reviewed supported a more negative attitude toward cancer than exhibited by either the normative group or the occupational health nurses in this study. Since this study was limited to one geographic location, a population of occupational health nurses in a different geographical area should be studied and the results compared to further establish reliability.
A plasminogen activator is induced during goldfish optic nerve regeneration.
The use of purified piscine plasminogen in a chromogenic solution assay enabled us to detect plasminogen activator (PA) activity in crude homogenates of goldfish optic nerve following nerve injury. In contrast, no activity was detected in the homogenates of uninjured nerve. Under conditions allowing regeneration of the optic axons (optic nerve crush), PA activity peaked 8 days after crush, and decreased to undetectable levels by 60 days. Under conditions allowing only degeneration of the axons (enucleation), the activity peaked at 8 days but decreased more rapidly. Casein zymography of samples after fractionation in SDS-PAGE showed that PA activity migrated as a doublet at Mr = 60-65 kd. Using this assay, activity was also observed in uninjured control nerves. This plasminogen-dependent activity migrated as three bands of higher molecular weight (Mr = 75, 95 and 120 kd) and was undetectable in solution assays of unfractionated extracts, suggesting complex formation with an inhibitor(s). Fibrin overlay assay of retinal explants and isolated primary cells in culture suggest that the goldfish PA is associated with the glial cells of the goldfish visual pathway.
Maintenance and use of F9 teratocarcinoma cells.
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Fractionation of heparin by chromatography on a tissue plasminogen activator-Sepharose column.
Heparin stimulates the activity of tissue plasminogen activator (t-PA) and binds to t-PA. To study this interaction, a complex between t-PA and N-acetylated heparin was formed and then linked to Sepharose. This procedure selectively links the t-PA to the column because the acetylated heparin has no free amino groups. The procedure also protects the heparin-binding site(s) on the enzyme during coupling to the matrix. The t-PA column separates heparin into two fractions, one with low affinity for t-PA and one with high affinity. Both fractions of heparin effectively accelerate inactivation of thrombin by antithrombin III. However, the fractions differ in their ability to stimulate t-PA: the low-affinity heparin has no effect on the activity of t-PA, whereas the high-affinity heparin enhances this activity. These heparin fractions will be useful in characterizing the biochemical basis and physiological consequences of the heparin--t-PA interaction.
Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation.
The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.
Shear bond strengths to dentin and fluoride release from fluoride-containing liners.
The purpose of this study was to determine the shear bond strengths of five commercially available fluoride-containing restorative systems to dentin and to evaluate the fluoride release from them. The five systems evaluated were: (A) Vitrabond/Silux; (B) Zionomer Powder Liquid/Perfection; (C) Zionomer Paste Paste/Perfection; (D) TimeLine/Prisma-Fil; and (E) GC Dental Cement/Fuji II. Eighteen test specimens were prepared on the dentin surfaces of extracted human permanent first and second molars ground on 600 grit SiC paper with each system. The bonded cylinders were removed from the assembly apparatus 15 minutes after cure, stored in physiological saline at 37 degrees C for 24 hours and subjected to a shear load at 0.5 mm/min. Five cylindrical specimens were prepared for each system, the discs were suspended in 25 microL glass distilled water and the fluoride release (microgram F/mm2) determined at daily intervals for 15 days and then after 30 and 90 days. The data were analyzed by a one-way analysis of variance and Duncan's multiple range test. The following shear bond strengths (Mean +/- SD) were recorded (MPa): A: 8.86 +/- 1.49; B: 4.24 +/- 1.23; C: 4.41 +/- 1.39; D: 3.85 +/- 1.60; E: 4.81 +/- 1.21. With all the systems, the fluoride release dropped sharply during the first 7 days and then remained constant. The shear bond strength and the fluoride release from Vitrabond/Silux were significantly greater than that of the other systems.
HIV-antibody testing in a multispecialty group practice.
The authors reviewed the trends in HIV-antibody testing at a multispecialty group practice with more than 280 physicians. Based on a chart review of 243 randomly selected patient records, they judged 38% of the tests ordered to be for low-risk individuals. Forty-five percent of the records documented the patient's informed consent before testing, and only 15% noted that patient education or counseling had been given.
Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator.
The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.
Structural domains of human tissue-type plasminogen activator that confer stimulation by heparin.
Heparin has been shown recently to stimulate the activity of human tissue-type plasminogen activator (t-PA). To investigate this effect further, mutant proteins lacking various domains of t-PA were screened for the ability to be stimulated by heparin. Those mutants harboring either the finger domain or the 2nd kringle were found to have enhanced enzymatic activity in the presence of heparin. Only mutants containing these structures would bind to heparin-agarose beads; monoclonal antibodies directed against these domains blocked binding. The stimulatory effect of heparin was more pronounced in finger-containing mutants than kringle-2 proteins. Earlier results had localized the fibrin-binding domains to the same two structures. Unlike heparin, the 2nd kringle was shown to be more important than the finger for fibrin stimulation. Our results have implications for producing recombinant t-PA variants for use in thrombolytic therapy.
Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes.
The translational activation of dormant tissue-type plasminogen activator mRNA during meiotic maturation of mouse oocytes is accompanied by elongation of its 3'-poly(A) tract. Injected RNA fragments that correspond to part of the 3'-untranslated region (3'UTR) of this mRNA are also subject to regulated polyadenylation. Chimeric mRNAs containing part of this 3'UTR are polyadenylated and translated following resumption of meiosis. Polyadenylation and translation of chimeric mRNAs require both specific sequences in the 3'UTR and the canonical 3'-processing signal AAUAAA. Injection of 3'-blocked mRNAs and in vitro polyadenylated mRNAs shows that the presence of a long poly(A) tract is necessary and sufficient for translation. These results establish a role for regulated polyadenylation in the post-transcriptional control of gene expression.
Differentiation-responsive elements in the 5' region of the mouse tissue plasminogen activator gene confer two-stage regulation by retinoic acid and cyclic AMP in teratocarcinoma cells.
F9 cells induced to differentiate with retinoic acid (RA) increase transcription of the tissue plasminogen activator (t-PA) gene. Further treatment of these cells with cyclic AMP (cAMP) results in an additional stimulation of t-PA gene transcription. To investigate the mechanism of this two-stage regulation, 4 kilobase pairs (kbp) of 5'-flanking sequence from the murine t-PA gene was isolated. Two major start sites for transcription were found, neither of which depended on a classical TATA motif for correct initiation. By using transient transfection assays, it was determined that 4-kbp of flanking sequence could confer on reporter genes the same two-stage differentiation-specific expression as was observed for the endogenous t-PA gene. Deletion analyses of this 4-kbp fragment showed that 190 bp of flanking sequence was sufficient to bestow the same degree of two-stage regulation on reporter gene constructs. Within this region of DNA, sequence analysis revealed a possible cAMP regulatory element, a CTF/NF-1 recognition sequence, two potential Sp1 sites, and five potential binding sites for transcription factor AP-2. The deletion experiments, coupled with the positions of these potential cis-acting elements, suggest that multiple transcription factors, including those that bind to cAMP regulatory element, CTF/NF-1, Sp1, and AP-2 sites, may be involved in regulation of the t-PA gene during F9 cell differentiation.
Antisense RNA directed against the 3' noncoding region prevents dormant mRNA activation in mouse oocytes.
Primary mouse oocytes contain untranslated stable messenger RNA for tissue plasminogen activator (t-PA). During meiotic maturation, this maternal mRNA undergoes a 3'-polyadenylation, is translated, and is degraded. Injections of maturing oocytes with different antisense RNA's complementary to both coding and noncoding portions of t-PA mRNA all selectively blocked t-PA synthesis. RNA blot analysis of t-PA mRNA in injected, matured oocytes suggested a cleavage of the RNA.RNA hybrid region, yielding a stable 5' portion, and an unstable 3' portion. In primary oocytes, the 3' noncoding region was susceptible to cleavage, while the other portions of the mRNA were blocked from hybrid formation until maturation occurred. Injection of antisense RNA complementary to 103 nucleotides of its extreme 3' untranslated region was sufficient to prevent the polyadenylation, translational activation, and destabilization of t-PA mRNA. These results demonstrate a critical role for the 3' noncoding region of a dormant mRNA in its translational recruitment during meiotic maturation of mouse oocytes.