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Biomedical subjects

S Strickland

Publications and source records attributed to S Strickland.

At least 55 records · Page 3Linked to original sources
PubMed1994

The mouse tissue plasminogen activator gene 5' flanking region directs appropriate expression in development and a seizure-enhanced response in the CNS.

Tissue plasminogen activator (t-PA) is a secreted serine protease implicated in multiple aspects of development. In the adult rat brain, transcription of t-PA is an immediate-early response in the hippocampus following treatments that induce neuronal plasticity. To study the sequence elements that govern transcription of this gene, in situ analysis was used to define t-PA's temporal and spatial expression pattern in midgestation embryos. Transgenic mice were then generated carrying t-PA 5' flanking sequences linked to the E. coli lacZ gene. Constructs containing 4 kb of the flanking sequences (4.0TAMGAL) confer beta-galactosidase activity mostly to the same tissues that exhibit high levels of t-PA mRNA by in situ analysis. In 4.0TAMGAL embryos from embryonic day 8.5 (E8.5) to 13.5 (E13.5), the majority of expression observed is localized to neural ectoderm-derived tissues. beta-galactosidase activity is first detected in restricted neuromeres in the midbrain and diencephalon, at E8.5 and E9.5 respectively. At E10.5, transgene expression is observed in neural crest-derived cranial nerves and dorsal root ganglia, but not placode-derived cranial nerves. From E10.5 to E13.5, beta-galactosidase activity is observed in postmitotic neurons of the midbrain, spinal cord, neural retina and the developing olfactory system. beta-galactosidase activity is also detected in areas undergoing tissue remodeling such as the pinna of the ear, whisker follicles and the limbs. In adult mice, lacZ is expressed in the hippocampus and this expression was found to be enhanced upon seizure in the giant pyramidal neurons of CA3. These results reinforce the concept that t-PA plays a role in neurogenesis and morphogenesis, and identifies the promoter region that directs its transcriptional regulation both in development and in the CNS.

Animals↗
PubMed1994

Aprotinin, a Kunitz-type protease inhibitor, stimulates skeletal muscle differentiation.

Aprotinin, a Kunitz-type inhibitor of serine proteases, stimulates myotube formation by mouse G8-1 and C2C12 skeletal muscle myoblasts. This stimulation of morphological differentiation is accompanied by accumulation of myogenin transcripts and production of muscle-specific proteins. In contrast, active TGF beta prevents differentiation of G8-1 and C2C12 myoblasts. When active TGF beta and aprotinin are both added to myoblast cultures, differentiation is inhibited, suggesting the active growth factor acts downstream of the protease inhibitor. TGF beta is found in serum as a latent, dimeric propolypeptide that is cleaved by limited proteolysis to release the biologically active carboxy-terminal dimer. To address the possibility that aprotinin may effect myogenesis by inhibiting proteolytic activation of latent TGF beta, levels of the endogenous growth factor were measured in differentiating myoblast cultures. Latent TGF beta is rapidly depleted from control cultures within 24 hours of plating, but remains relatively stable in aprotinin-treated cultures. Consistent with this, aprotinin-treated cultures have reduced levels of active TGF beta. These data indicate that Kunitz-domain containing protease inhibitors may help orchestrate the onset of myogenesis, possibly by regulating the activity of TGF beta-like molecules.

Amyloid beta-Protein Precursor↗
PubMed1993

Creating maternal effect mutations in transgenic mice: antisense inhibition of an oocyte gene product.

Gene products present in mouse oocytes direct development until the two-cell stage and may be important in later development. Here, we demonstrate that expression of a specific maternal protein can be disrupted in mouse oocytes using transgenic antisense RNA technology. An oocyte-specific promoter (mZP3) was utilized to express antisense RNA directed against maternal mRNA encoding tissue-type plasminogen activator (tPA). Antisense expression results in reduced levels of tPA mRNA and enzyme activity in mouse oocytes. We also provide evidence for a novel mechanism of antisense-mediated translational inhibition, whereby the cytoplasmic polyadenylation of maternal tPA mRNA is altered. This strategy should prove applicable to functional studies of other murine maternal mRNAs in an in vivo environment.

Animals↗
PubMed1993

Preimplantation mouse embryos express a cell surface receptor for tissue-plasminogen activator.

The serine protease tissue-plasminogen activator (t-PA) has previously been shown to be intracellular in mouse secondary oocytes and extracellular in fertilized eggs. Here we demonstrate that extracellular t-PA activity is bound to the surface of the fertilized egg. The level of t-PA activity associated with preimplantation mouse embryos decreases in the 2-cell stage embryo, then increases in 4-cell and morula stage embryos. However, morulae grown in culture from fertilized eggs lack t-PA activity but are able to bind exogenously added mouse t-PA. Additionally, northern analysis indicates that preimplantation embryos do not contain detectable levels of t-PA mRNA. Therefore, the enzyme activity associated with 4-cell and morula stage embryos in vivo is derived from t-PA present in the oviduct lumen that binds the embryo, and not from protein produced from translation of embryonic mRNA. The binding activity is species and protein specific in that neither mouse urokinase-type plasminogen activator (u-PA) nor human t-PA bind to cultured morulae. Furthermore, binding activity is dose-dependent and saturable, and does not require the active site of t-PA. These data indicate that a cell surface-specific t-PA-binding activity exists in the preimplantation mouse embryo and may localize function and concentrate the proteolytic activity of t-PA in early mouse development.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

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.

AIDS Serodiagnosis↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Adult↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Angiotensin III↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Acrylic Resins↗
PubMed1990

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.

AIDS Serodiagnosis↗