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Biomedical subjects

A Sandra

Publications and source records attributed to A Sandra.

At least 19 recordsLinked to original sources
PubMed2002

Structure-function relationships of insulin-like growth factor binding protein 6 (IGFBP-6) and its chimeras.

Insulin-like growth factor binding protein 6 (IGFBP-6) is a high-affinity IGFBP with substantially greater affinity for insulin-like growth factor-II (IGF-II) than IGF-I. IGFBP-6(3) is a chimera which has a 20 amino acidC -terminal portion of IGFBP-6 switched with the homologous area of IGFBP-3, P3. Unlike IGFBP-4(3), in which the P3 region was exchanged for the homologous region of IGFBP-4 (P4), IGFBP-6(3) does not bind to endothelial cells. Double mutations were made with the P3 region exchanged as well as a second area differing from IGFBP-3 to form IGFBP-6(3)A and IGFBP-6(3)B, by replacing this area with the homologous region of IGFBP-3. Neither [(125)I]IGFBP-6(3)A nor IGFBP-6(3)B specifically bound to endothelial cells. However, each double mutant competed for [(125)I]IGFBP-3 binding to cultured cells. In the perfused heart, transendothelial transport of IGFBP-6 and IGFBP-6(3) was only 25% of similar transendothelial transport of perfused IGFBP-3. We conclude that chimeras of IGFBP-6 and IGFBP-3(6) clearly differ from IGFBP-4(3) in their ability to bind specifically to endothelial cells and in their capacity to undergo transendothelial transportation in the perfused heart.

Amino Acid Sequence↗
PubMed2001

Vascular endothelial growth factor and basic fibroblast growth factor differentially modulate early postnatal coronary angiogenesis.

The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary growth that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar growth, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles (<15 microm) was markedly reduced, whereas the percentage of large arterioles (26 to 50 microm) more than doubled. Thus, inhibition of both growth factors negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary growth, (2) bFGF facilitates arteriolar growth, and (3) the two growth factors interact to establish the normal hierarchy of the arteriolar tree.

Animals↗
PubMed2001

Distribution of chimeric IGF binding protein (IGFBP)-3 and IGFBP-4 in the rat heart: importance of C-terminal basic region.

IGF binding proteins-3 and -4, whether given in the perfused rat heart or given iv in the intact animal, cross the microvascular endothelium of the heart and distribute in subendothelial tissues. IGF binding protein-3, like IGF-I/II, localizes in cardiac muscle, with lesser concentrations in CT elements. In contrast, IGFBP-4 preferentially localizes in CT. In this study, chimeric IGF binding proteins were prepared in which a basic 20-amino-acid C-terminal region of IGF binding protein-3 was switched with the homologous region of IGF binding protein-4, and vice-versa, to create IGF binding protein-3(4) and IGF binding protein-4(3). Perfused IGF binding protein-3(4) behaved like IGF binding protein-4, localizing in connective tissue elements, whereas IGF binding protein-4(3) now localized in cardiac muscle at concentrations identical to perfused IGF binding protein-3. To determine whether these small mutations altered the affinity of the chimera for cells, the ability of (125)I-IGF binding protein-3(4) and (125)I-IGF binding protein-4(3) to bind to microvascular endothelial cells was determined and compared with IGF binding protein-3. IGF binding protein-3(4) retained 15% of the binding capacity of IGF binding protein-3, whereas IGF binding protein-4(3) bound to microvessel endothelial cells with higher affinity and greater total binding than that of IGF binding protein-3. We conclude that small changes in the C-terminal basic domain of IGF binding protein-3 and the corresponding region of IGF binding protein-4 can alter their affinity for cultured cells and influence their tissue distribution in the rat heart.

Amino Acid Sequence↗
PubMed2000

Interinstitutional study to compare the effectiveness of a radiology-anatomy module of instruction.

RATIONALE AND OBJECTIVES: The purpose of this study was to validate the effectiveness of a proven radiology-anatomy instructional module during I st-year gross anatomy courses at Emory University College of Medicine and the University of Iowa College of Medicine. MATERIALS AND METHODS: This prospective study involved 108 Emory students and 177 Iowa students. The instructional content was the same at both institutions. Each student was randomly assigned into one of three groups at each institution, and each group received a unique, randomized, five-item pretest. All students were posttested as part of their gross anatomy laboratory examination, and the posttests consisted of all 15 items used in the three five-item pretests. RESULTS: No statistically significant pretest effects were demonstrated by t tests. Posttest performances across items ranged from 73% to 96% correct for Emory students and 67% to 98% for Iowa students. Performance levels on the posttests were significantly higher than on pretests, and few significant differences were found in the performance of the two populations. CONCLUSION: The radiology-anatomy instructional module integrated into the gross anatomy courses for 1st-year Emory University and University of Iowa students was not instructor or institution dependent.

Anatomy↗
PubMed1999

Vascular endothelial growth factor expression coincides with coronary vasculogenesis and angiogenesis.

Vascular endothelial growth factor (VEGF) plays an important role in early embryonic vasculogenesis. To establish its temporal expression and localization in the heart during development, we studied rat hearts from the first embryonic day (E) of myocardial vascular tube formation through the early postnatal period. Ventricular VEGF immunoreactivity was noted in the epicardium and the thin underlying myocardium in E10 ventricles. During the earliest stages of vascularization (E13-E16) immunoreactivity was highest in the compact myocardium nearest the epicardium, and subsequently (E18 and thereafter) became more evenly distributed transmurally. By birth (E22) immunoreactivity was most intense around microvessels. Similarly, VEGF mRNA localization, demonstrated by in situ hybridization, was initially highest near the epicardium and then became more evenly distributed transmurally by late gestation. Within the interventricular septum, the highest expression occurred in the middle of the wall where it correlated with the greatest vascularization. Northern blot analysis showed that from E12 through the first 10 days of postnatal life, VEGF was two to three times higher than in the adult. Western blot analysis showed that VEGF tended to be higher in the atria than the ventricles, and negligible in the outflow tract. Our data indicate that VEGF localization and expression 1) correspond to the pattern of vascularization in the embryonic/fetal heart, and 2) remain high during the early postnatal period when capillary proliferation is high. Because VEGF is stimulated by hypoxia, its preferential mRNA expression near the epicardium, that is, farthest from the ventricular lumen and the O2 source, fits with the hypothesis that a hypoxic gradient is a driving force in the transmural vascularization process.

Age Factors↗
PubMed1999

Decreased expression of the c-kit receptor is associated with increased apoptosis in subfertile human testes.

OBJECTIVE: To examine the expression of the c-kit receptor and its ligand, stem cell factor, and their possible relation with apoptosis in infertile men. DESIGN: Prospective laboratory study. SETTING: Urology laboratory in a university hospital. PATIENT(S): Men undergoing testicular biopsy during an investigation of subfertility. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Expression of the c-kit receptor protein, stem cell factor, and apoptosis in the testes. RESULT(S): The c-kit receptor was strongly present in Leydig cells and type A spermatogonia of normal testes, with decreased staining in Leydig cells and type A spermatogonia of testes with maturational arrest, and staining in only Leydig cells of Sertoli cell-only specimens. Stem cell factor was demonstrated in Leydig cells and Sertoli cells in all specimens. Western blotting demonstrated the 150-kd c-kit protein in the normal testes and the testes with maturational arrest, but not in the testes with the Sertoli cell-only pattern. Stem cell factor was expressed in all specimens, with a protein size of 45 kd. Increased apoptosis was demonstrated in type A spermatogonia and spermatocytes of tissue with maturational arrest compared with normal testicular tissue. CONCLUSION(S): C-kit receptor expression is decreased in subfertile testicular tissue compared with normal testicular tissue. Stem cell factor expression is present in Leydig cells and Sertoli cells. Increased apoptosis is seen in tissue with maturational arrest compared with normal tissue.

Adult↗
PubMed1999

Temporal expression of inducible nitric oxide synthase in mouse and human placenta.

The aim of this study is to investigate the changes in expression and activity of inducible nitric oxide synthase (iNOS) in the developing murine embryo and mouse and human placenta. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and in-situ hybridization (ISH) we identified iNOS mRNA in mouse placenta at 9.5, 12, 14, 16, 18 and 20 days post coitum. Northern blot analysis demonstrated that the quantity of murine iNOS transcript was expressed at a stable level between days 12-20 although the level of calcium-independent NOS activity declined with advancing gestation. RT-PCR detected iNOS-specific mRNA in murine embryonic stem cells, but not in embryos at later stages (4-cell or blastocyst). ISH failed to show iNOS-specific mRNA in either murine placenta or the underlying myometrium on day 7, but did so in the trophoblast by day 9.5. Later in gestation, extensive labelling was observed in both spongiotrophoblast and trophoblast giant cells. iNOS mRNA was also detected both in immature human placentae (16-18 weeks) and at term, predominantly in syncytiotrophoblasts and placental artery smooth muscle. In conclusion, iNOS is constitutively expressed in mouse and human placenta at a time and in a location that suggests a role in placentation.

Animals↗
PubMed1999

Fetal hemorrhage and platelet dysfunction in SLP-76-deficient mice.

The adapter protein SLP-76 is expressed in T lymphocytes and hematopoietic cells of the myeloid lineage, and is known to be a substrate of the protein tyrosine kinases that are activated after ligation of the T-cell antigen receptor. Transient overexpression of SLP-76 in a T-cell line potentiates transcriptional activation after T-cell receptor ligation, while loss of SLP-76 expression abrogates several T-cell receptor-dependent signaling pathways. Mutant mice that lack SLP-76 manifest a severe block at an early stage of thymocyte development, implicating SLP-76 in signaling events that promote thymocyte maturation. While it is clear that SLP-76 plays a key role in development and activation of T lymphocytes, relatively little is understood regarding its role in transducing signals initiated after receptor ligation in other hematopoietic cell types. In this report, we describe fetal hemorrhage and perinatal mortality in SLP-76-deficient mice. Although megakaryocyte and platelet development proceeds normally in the absence of SLP-76, collagen-induced platelet aggregation and granule release is markedly impaired. Furthermore, treatment of SLP-76-deficient platelets with collagen fails to elicit tyrosine phosphorylation of phospholipase C-gamma2 (PLC-gamma2), suggesting that SLP-76 functions upstream of PLC-gamma2 activation. These data provide one potential mechanism for the fetal hemorrhage observed in SLP-76-deficient mice and reveal that SLP-76 expression is required for optimal receptor-mediated signal transduction in platelets as well as T lymphocytes.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Effects of TGFbeta2 on collagen synthesis in cultured normal and wounded fetal mouse palates.

OBJECTIVE: It has been demonstrated in a number of models that fetal wounds heal with little or no scar. Since collagen is an integral part of the extracellular matrix in adult scar formation, we studied the synthesis and localization of collagen in an in vitro mouse palate model for fetal wound healing. METHODS: Palates, dissected from fetal mice at 15, 16, and 17 days of gestation and from newborn mice, were cultured in medium containing serum (for 8 hours); this was followed by culture in serum-free medium (for 12 hours). One-half of the samples from each age group were wounded in the midline. All samples were placed in serum-free medium containing 20 microCi/mL 3H-proline for 8 hours. In addition, palates from 15-day gestation and from newborn mice were also incubated with transforming growth factor TGF-beta2 (10 ng/mL). Palates were washed with saline, homogenized, and radioactivity was counted. Proline uptake was calculated for each sample as counts per milligram of protein and was subjected to statistical analysis (three-way analysis of variance). Samples of the homogenate were subjected to sodium dodecyl sulfate-gel electrophoresis and Western blotting in order to determine the types of collagen that were synthesized. Immunohistochemical localization of collagen types I, III, and VI was carried out on paraffin-embedded samples from each group. RESULTS: There were no significant differences in proline uptake between wounded mouse palates and nonwounded mouse palates at any age, and there was no histological evidence of regeneration of the palate at the site of the wound. Proline uptake was significantly greater in untreated wounded palates at 15 days' gestation than it was in newborns. After treatment with TGF-beta2, proline uptake was significantly greater in both wounded and nonwounded palates in the newborn group and had no effect on collagen synthesis in palates from 15-day gestation animals. Collagen types I and III were localized in histological specimens using immunohistochemistry and on nitrocellulose using Western blotting. No type VI collagen was demonstrated by Western blotting, but it was localized around blood vessels and on basement membranes using immunohistochemistry. CONCLUSION: Treatment with TGF-beta2 significantly increased collagen synthesis, as assessed by 3H-proline uptake, in cultured palates from newborn mice as compared with palates from untreated newborn mice and from both treated and untreated palates of 15-day gestation mice. These data suggest a differential response to TGF-beta2 by mouse palates as a function of fetal development.

Analysis of Variance↗
PubMed1999

Migration and ultrastructural localization of the c-kit receptor protein in spermatogenic cells and spermatozoa of the mouse.

PURPOSE: The c-kit receptor is a proto-oncogene important in germ cell migration and maturation and has also been demonstrated on the acrosomal region of mature sperm. The purpose of the present study was to examine the ultrastructural location of the c-kit receptor in mouse testis and sperm. MATERIALS AND METHODS: Testis and sperm from mature male mice were examined for the c-kit receptor utilizing electron microscopy and Western blot analysis techniques. Thin sections of mouse testis and sperm were stained with immunogold-labeled anti-c-kit antibodies. The protein from these testes and sperm was also utilized for Western blot analysis. RESULTS: The c-kit protein was localized within the mouse testes to the type A spermatogonia, the round spermatids, and the mature testicular spermatozoa. The c-kit receptor was noted to migrate from the lumen of the acrosomal vesicles in the early spermatids to the plasma membrane of the late spermatids. It was also noted in the acrosomal region of the testicular spermatozoa, as well as the sperm from the epididymis. Sperm undergoing the acrosome reaction demonstrated association of the c-kit receptor with the plasma membrane of the acrosome, but not on the acrosomal membrane itself. Western blot analysis demonstrated protein bands of 150 kDa in testis and intact sperm. CONCLUSIONS: The present study confirms the presence of the c-kit receptor in mouse testis and sperm. It also demonstrates that this receptor is localized to the region of the developing acrosome.

Acrosome Reaction↗
PubMed1999

Development of an immunocontraceptive vaccine. Current status.

Development of an effective and modern contraceptive vaccine is a key factor in the global issue of regional population growth as well as agricultural, medical, economic and social development. A review was done of the current medical literature concerning development of an immunocontraceptive vaccine and relative molecular biology technology. Various approaches have been taken to identify candidate-specific antigens for immunocontraceptive development, such as sperm, zona pellucida and hormonal antigens. Suppressed fertility and the reversibility of these effects on mammalian species, including humans, have been demonstrated. The successful results obtained so far support the continued investigation for an effective immunocontraceptive vaccine.

Animals↗
PubMed1998

Early coronary angiogenesis in response to thyroxine: growth characteristics and upregulation of basic fibroblast growth factor.

Although a substantial coronary angiogenesis occurs after thyroid hormone treatment, its regulation and relationship to cardiac hypertrophy are not understood. This study was designed to determine (1) the onset of capillary proliferation, (2) the sites of capillary proliferation, and (3) whether basic fibroblast growth factor (bFGF) upregulation occurs in response to thyroxine administration. Male Sprague-Dawley rats were injected daily with L-thyroxine (T4, 0.2 mg/kg s.c.). Bromodeoxyuridine labeling of capillary endothelial cells increased during the first 24 hours of treatment and peaked after 2 days of treatment. Northern blot analysis revealed a slight increase in bFGF mRNA during this period, followed by a doubling of expression by 48 hours, at which time bFGF protein was also increased. In situ hybridization, used to localize bFGF mRNA, showed an increase in transcripts within 24 hours after T4. This enhancement was uniform in the epimyocardium and endomyocardium. Histochemical analysis (double staining for alkaline phosphatase and dipeptidyl peptidase) of frozen sections, used to discriminate capillary profiles as arteriolar and venular, respectively, showed that growth occurred in the latter, since the percentage of capillary profiles positive for dipeptidyl peptidase was higher than the control value after 4 days of T4 administration. These data indicate that in the thyroxine model of cardiac hypertrophy (1) capillary DNA synthesis occurs after a single injection of thyroxine, (2) capillary growth coincides with an upregulation in bFGF mRNA and increase in bFGF protein, and (3) proliferation occurs in the venular capillaries.

Animals↗
PubMed1998

The c-kit receptor and its possible signaling transduction pathway in mouse spermatozoa.

The presence and role of the c-kit protein was investigated in the mature sperm of the mouse. The c-kit monoclonal antibody (mAb) ACK2 reacted specifically with the acrosomal region and the principal piece of fixed noncapacitated sperm but did not react with the acrosome region in acrosome-reacted sperm. ACK2 significantly inhibited the acrosome reaction; this inhibition was relieved by the calcium ionophore A23187. The kit ligand stem cell factor (SCF) significantly increased the percentage of sperm undergoing acrosome reaction. This increase was partially inhibited by the calcium channel inhibitor (verapamil), the PI3k inhibitor (wortmannin), and the PLC inhibitor (U-73122). ACK2 predominantly recognized c-kit proteins of 33, 48, and 150 kDa by Western blotting of mouse sperm extracts. The 48- and 150-kDa protein bands were released into the media and tyrosine autophosphorylated at low basal levels during acrosome reaction. On stimulation with SCF, the level of c-kit phosphorylation increased significantly. These findings suggest that c-kit is present in mature sperm, and its binding to SCF may result in the activation of PLC gamma 1 and PI3K, leading to receptor autophosphorylation, and ultimately may play a role in capacitation and/or the acrosome reaction.

Animals↗
PubMed1998

Direct evidence for nitric oxide synthase in vagal afferents to the nucleus tractus solitarii.

The anatomical relationship between vagal afferents and brain nitric oxide synthase containing terminals in the nucleus tractus solitarii was studied by means of anterograde tracing combined with immunocytochemistry and immuno-electron microscopy. Biotinylated dextran amine was injected into the nodose ganglion with a glass micropipette. Four to eight days following the injection, regions of the nucleus tractus solitarii containing biotinylated dextran amine-labelled vagal afferents and those containing nitric oxide synthase-immunopositive terminals were congruent. Many neurons exhibiting nitric oxide synthase immunoreactivity were found within the biotinylated dextran amine-containing terminal field. However dense labeling of terminals with biotinylated dextran amine precluded determination if the terminals were nitric oxide synthase-immunoreactive. Therefore, we combined degeneration of vagal afferents after removal of one nodose ganglion with nitric oxide synthase immuno-electron microscopy. Axon terminals that possessed characteristic vesicle clusters and were partially or completely engulfed by glial processes were identified as degenerating vagal afferents. Degenerating axon terminals comprised 38% of the total axon terminals in the nucleus tractus solitarii in a sample of sections; and of the degenerating axon terminals, 67% were nitric oxide synthase-immunoreactive. Nitric oxide synthase immunoreactivity was present in 41% of the non-degenerating axon terminals. Prominent staining of dendrites for nitric oxide synthase immunoreactivity indicated that much of the nitric oxide synthase in the nucleus tractus solitarii is not derived from peripheral afferents. Of the total number of dendritic profiles sampled, half were nitric oxide synthase-immunoreactive. Our data support the hypothesis that nitric oxide or nitric oxide donors may be present in primary vagal afferents that terminate in the nucleus tractus solitarii. While this study confirms that vagal afferents contain brain nitric oxide synthase, it demonstrates for the first time that the majority of nitric oxide synthase immunoreactivity in the nucleus tractus solitarii is found in intrinsic structures in the nucleus. In addition, our data show that second or higher order neurons in the nucleus tractus solitarii may be nitroxidergic and receive both nitroxidergic and non-nitroxidergic vagal input.

Afferent Pathways↗
PubMed1998

Infused IGF-I/IGFBP-3 complex causes glomerular localization of IGF-I in the rat kidney.

Insulin-like growth factor I (IGF-I) increases renal blood flow, glomerular filtration rate (GFR), and proximal tubule reabsorption of phosphate in humans and rodents. The biological effects of IGF-I are likely to be influenced by cellular localization of IGF-I within the kidney. We therefore tested whether the renal localization of infused IGF-I could be altered if given with selected IGF-binding proteins (IGFBPs). Rats were treated with intravenous injections of 125I-labeled IGF-I, 125I-IGFBP-3, or 125I-IGFBP-4 alone or with complexes of 125I-IGF-I and IGFBP-3 or IGFBP-4. The cellular localization of IGF and the IGFBP within the kidney was then determined. 125I-IGF-I, 125I-IGFBP-4, and 125I-IGF-I/IGFBP-4 complexes were found almost exclusively in vacuolar structures (endosomes) of proximal renal tubules. In contrast, about one-third of renal 125I-IGFBP-3 and 125I-IGF-I/IGFBP-3 was localized to glomeruli. When 125I-IGF-I was given alone, 3% was found in glomeruli and 89% in proximal tubules. When given as 125I-IGF-I/IGFBP-3, 29% was in glomeruli and 65% in proximal tubules. We conclude that the cellular localization of IGF-I within the kidney can be directed to glomerular elements if the IGF-I is given with IGFBP-3.

Animals↗
PubMed1997

Up-regulation of nitric oxide synthase and its mRNA in vagal motor nuclei following axotomy in rat.

Effects of vagotomy on nitric oxide synthase (NOS) protein and mRNA levels in the dorsal motor nucleus of vagus (DMV) and nucleus ambiguus (NA) of rats were examined by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining, brain NOS (bNOS) immunostaining and in situ hybridization. NADPH-d staining and bNOS immunoreactivity increased in neurons of the ipsilateral DMV and NA 5, 10, and 20 days after vagotomy. These changes were not observed in unoperated or sham-operated rats. In situ hybridization showed that bNOS mRNA levels were also elevated in neurons of DMV and NA on the operated side. Our results suggest that transection of vagal efferents up-regulates bNOS and its mRNA expression in the DMV and NA.

Animals↗