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Biomedical subjects

A Sandra

Publications and source records attributed to A Sandra.

At least 37 records · Page 2Linked to original sources
PubMed1997

Localization and expression of the c-kit receptor protein in human and rodent testis and sperm.

OBJECTIVES: To examine the localization and expression of the c-kit receptor protein in the testes of the mouse, rat, and human, and then compare these among the three species. METHODS: Testis tissue from all three species was obtained through biopsy or orchiectomy. Immunohistochemistry was used for the localization, using a monoclonal antibody to the c-kit receptor. The expression of the c-kit receptor protein was examined in the testes and sperm with Western blot analysis. RESULTS: Localization was noted in the early spermatogenic cells, most likely type A spermatogonia, as well as in the acrosomal region of more mature germ cells, such as the round spermatids. The c-kit receptor was localized in analogous sites in all three species. The Western blot data revealed testicular expression of the c-kit receptor protein in all three species as well. Similar bands were recognized on the Western blots of all three species in testes at approximately 75 kDa and approximately 90 kDa, and sperm at approximately 90 kDa only. CONCLUSIONS: The c-kit receptor protein is expressed in the early spermatogenic cells, as well as the later stages of spermatogenesis, specifically, the acrosomal granules of the round spermatids, and the acrosomal region of testicular spermatozoa, in the mouse, rat, and human. All three species exhibit similar expression of the c-kit receptor protein in both testis and sperm, although to a varying degree. We believe that these observations allow direct valid comparisons concerning the expression of the c-kit receptor to be made cautiously to the human condition from experimental data obtained from rodents.

Animals↗
PubMed1997

Expression and function of the c-kit proto-oncogene protein in mouse sperm.

The presence and role of the c-kit protein were examined in mature sperm of the mouse. Monoclonal antibodies (mAbs) against the c-kit protein were used to perform immunohistochemical staining, electron microscopy studies, and Western blot analysis. The acrosomal region of both fixed and unfixed noncapacitated sperm stained with the antibodies. No acrosomal staining was noted in acrosome-reacted (AR) sperm. Electron microscopy studies demonstrated immunogold label on the plasma membrane of the acrosome and confirmed the lack of binding following the acrosome reaction. Proteins corresponding to 33 kDa, 48 kDa, and 150 kDa were detected by the antibodies utilizing Western blot analysis. The 48-kDa and 150-kDa proteins were released into the media during sperm capacitation, and release from the acrosome was dependent upon the acrosome reaction. The mAbs significantly inhibited the acrosome reaction and increased sperm agglutination. Monoclonal antibody ACK1 significantly inhibited the motility of the sperm, whereas mAbs ACK2 and NCL-ckit did not. These results suggest that c-kit-related proteins are present in mature sperm and may play a role in capacitation and/or the acrosome reaction.

Acrosome↗
PubMed1997

Increased expression of nitric oxide synthase in the gracile nucleus of aged rats.

Aging is associated with disturbances in autonomic cardiovascular control. The purpose of this study was to test the hypothesis that changes in nitric oxide occur with aging in brainstem nuclei involved in autonomic cardiovascular control. The principal and unexpected finding in this study was that NADPH-diaphorase reactivity, which is considered a marker of neuronal nitric oxide synthase activity, was decidedly increased in the neuronal bodies of the gracile nucleus but decreased in the axons and axon terminals in old compared with young rats. In situ hybridization also revealed that nitric oxide synthase gene expression was enhanced predominantly in the gracile nucleus neurons of aged rats. The differences between the young and old rats were most dramatically evident in the gracile nucleus, but not evident in other brainstem nuclei. The significance of this finding as it might relate to autonomic or other specific neural dysfunction with aging is not evident at this time.

Aging↗
PubMed1996

Coronary vascularization during development in the rat and its relationship to basic fibroblast growth factor.

OBJECTIVE: Our overall aims were to elucidate the temporal and spatial sequence of coronary vascularization during development in the rat, and to determine whether basic fibroblast growth factor expression corresponds to any phase of the vascularization process. METHODS: Immunohistochemical, histochemical, morphometric and in situ hybridization analyses were performed on prenatal and postnatal hearts of various ages. RESULTS: Coronary vascularization, which begins at embryonic day 13 (E13) with blood island-like structures in the epicardium, progresses from this layer toward the endocardium as indicated by a transmural gradient of vascular volume throughout the ventricles. Vascular smooth muscle first appears in E17 hearts at the time a capillary-like plexus coalesces and penetrates the aorta to form the main coronary arteries. These vessels maintain an anastomatic morphology and must undergo subsequent remodeling in order to assume adult branching characteristics. The early postnatal period is characterized by development of the arterial tree and the enzymatic differentiation of the arteriolar and venular ends of the capillary bed. Although bFGF is expressed both prenatally and postnatally, the highest mRNA expression was noted during the early period of vascularization (E14 and E15), and the early neonatal period (1-6 days) which corresponds to a period of substantial microvascular growth. CONCLUSIONS: Coronary vascularization follows a temporal sequence which includes transmural expansion of the capillary bed, arteriolar formation subsequent to vascular penetration of the aorta, and postnatal growth, differentiation, and remodeling. Since high levels of bFGF expression are correlated with key time points in coronary vascular growth, bFGF may play an important role in this process.

Alkaline Phosphatase↗
PubMed1996

The role of transforming growth factor alpha in rat craniofacial development and chondrogenesis.

To explore the possible role of transforming growth factor alpha (TGF-alpha) in craniofacial development, its expression in the craniofacial region of rat embryos from embryonic day (d) 9 to d 20 was examined by in situ hybridisation and immunostaining. The TGF-alpha transcripts were first detected in the neural fold of embryonic d 9 and 10 embryos. In the craniofacial region, the TGF-alpha transcripts were not detected until embryonic d 16 in mesenchyme surrounding the olfactory bulb, within the olfactory bulb, the nasal capsule, vomeronasal organ, and vibrissal follicle. In addition, TGF-alpha message was detected in mesenchyme in the vicinity of Meckel's cartilage, and in the dental epithelium and lamina. This expression pattern of TGF-alpha transcripts persisted until embryonic d 17 but disappeared by d 18. The presence of TGF-alpha protein largely coincided with TGF-alpha message although, unlike the message, it persisted throughout later embryogenesis in the craniofacial region. The possible function of TGF-alpha in chondrogenesis was explored by employing the micromass culture technique. Cartilage nodule formation in mesenchymal cells cultured from rat mandibles in the presence of TGF-alpha was significantly inhibited. This inhibitory effect of TGF-alpha on chondrogenesis was reversed by addition of antibody against the EGF receptor, which crossreacts with the TGF-alpha receptor. The inhibitory effect of TGF-alpha on chondrogenesis in vitro was further confirmed by micromass culture using mesenchymal cells from rat embryonic limb bud. Taken together, these results demonstrate the involvement of TGF-alpha in chondrogenesis during embryonic development, possibly by way of a specific inhibition of cartilage formation from mesenchymal precursor cells.

Animals↗
PubMed1994

Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin-Darby canine kidney) cells. Evidence for the reverse action of a sphingomyelin synthase.

When N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-PA) is inserted into the plasma membrane of fibroblasts, it is metabolized by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -phosphatidylcholine (PC), and -phosphatidylethanolamine (PE) (Pagano, R. E., Longmuir, K. J., and Martin, O. C. (1983) J. Biol. Chem. 258, 2034-2040). In Madin-Darby canine kidney (MDCK) cells incubated at 10 degrees C with C6-NBD-PA, up to 70% of the newly synthesized C6-NBD-PC but no C6-NBD-PE could be depleted from the basolateral cell surface by the addition of bovine serum albumin to the medium. Preincubation of the cells with [3H]choline for 2 h at 37 degrees C prior to C6-NBD-PA addition at 10 degrees C labeled non-depletable C6-NBD-PC with a specific activity of > 10 times that of the depletable C6-NBD-PC on the basolateral cell surface, indicating that the latter had not been synthesized by the CDP-choline pathway. C6-NBD-DG could substitute for C6-NBD-PA as substrate for both intracellular and surface C6-NBD-PC synthesis. In addition, C6-NBD-PC synthesis on the cell surface was independent of the location of the C6-NBD-chain on the 1- or 2-position, indicating that the reaction occurred by transfer of phosphorylcholine. Using C6-NBD-ceramide, C6-NBD-sphingomyelin (SM) synthesis also was discovered on the basolateral but not on the apical cell surface. The conversion of PC plus ceramide to DG and SM on the basolateral MDCK cell surface suggests that the synthesis of C6-NBD-PC on this surface occurred via the reverse reaction of a SM synthase. Indeed, the surface C6-NBD-PC synthesis was reduced to 40-50% by addition of C6-NBD-ceramide or hydrolysis of cell surface SM by exogenous neutral sphingomyelinase. Since DG activates protein kinase C and ceramide indirectly inhibits this kinase but activates other kinase(s) and phosphatase(s), the phosphocholine transferase at the cell surface may have a regulatory role in signal transduction.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Transport of transferrin across the blood-thymus barrier in young rats.

The interaction of transferrin-peroxidase (Tf-HRP) with the capillary endothelium of the rat thymus was analyzed by diaminobenzidine (DAB) cytochemistry and electron microscopy. The thoracic aortas of young rats were cannulated and the upper bodies perfused with the Tf-HRP conjugate. In the thymus, plasmalemmal vesicles of the endothelium mediated the endocytosis and transport of Tf-HRP. Transcytosis of Tf-HRP appeared to occur by micropinocytosis, without morphological evidence of involvement by endothelial endosomes. DAB reaction product was commonly present in the subendothelial interstitial space and, in addition, was often localized in clathrin coated pits and vesicles in epithelial reticular cells that surround the thymic capillaries. In perfusions done at 4 degrees C, no binding of Tf-HRP to the lumenal membranes of capillaries was detected. The transport of Tf-HRP across the capillary endothelium in the thymus was not inhibited by competition with excess native transferrin; however, the uptake of Tf-HRP by epithelial reticular cells was completely inhibited by excess transferrin. Transferrin receptors were localized in the thymus by indirect immunocytochemistry using OX-26, a mouse anti-rat transferrin receptor monoclonal antibody. No transferrin receptors were detected on the capillary endothelium but diffuse reaction product was localized on the subjacent epithelial reticular cells. These results indicate that transport of Tf-HRP across the thymus capillary wall is independent of transferrin receptors at the level of the endothelial cells but that subsequent uptake of Tf-HRP by epithelial reticular cells is a transferrin receptor dependent process.

Age Factors↗
PubMed1993

Immunolocalization of GLUT-1 glucose transporter in rat skeletal muscle and in normal and hypoxic cardiac tissue.

We compared the expression and cell-type localization of GLUT-1 mRNA and protein between cardiac and skeletal muscle of normal rats. Also, since we recently showed that cardiac GLUT-1 is upregulated in rats exposed to hypobaric hypoxia, we examined the cellular localization of GLUT-1 in cardiac tissue of normal and hypoxic rats. Confocal light microscopy and double immunofluorescent labeling revealed intense localization of GLUT-1 around neurofilament immunoreactivity within gastrocnemius muscle consistent with the previously described localization of large amounts of GLUT-1 in perineurial sheaths of skeletal muscle. However, using the same methods, we were unable to visualize GLUT-1 adjacent to nerve fibers in numerous sections of right or left ventricles or atria. Compared with skeletal myoctes, however, GLUT-1 immunofluorescence among cardiomyocytes was much more intense, particularly along the plasma membrane and especially intercalated discs. GLUT-1 immunofluorescence was also seen within the walls of arterioles within the heart. The predominant localization of GLUT-1 expression to cardiomyocytes in heart tissue was confirmed by in situ mRNA hybridization to digoxigenin-conjugated GLUT-1 cDNA. Northern blot analysis demonstrated that GLUT-1 mRNA was increased severalfold in the cardiac tissues compared with skeletal muscle. Although we detected GLUT-1 protein by immunoblotting of detergent extracts of the heart, we could not detect GLUT-1 in similar extracts of skeletal muscle. The cell type distribution of GLUT-1 in hearts of hypoxic rats was not different by immunohistochemistry from normals. These data indicate that 1) the cell-type distribution of GLUT-1 in the heart differs markedly from that in skeletal muscle. GLUT-1 in cardiac tissue, unlike skeletal muscle, is predominantly expressed within myocytes. 2) Cardiac GLUT-1 is not located along nerve fibers. 3) GLUT-1 mRNA and protein levels in cardiac tissue are considerably greater than in skeletal muscle. 4) The hypoxia-induced increase in cardiac GLUT-1 that we previously reported must occur within cardiomyocytes.

Animals↗
PubMed1993

Receptor-mediated endocytosis of transferrin at the blood-brain barrier.

Rat brains were perfuse with a transferrin-peroxidase conjugate (Tf-HRP) to characterize morphologically the endocytic pathway of transferrin in blood-brain barrier endothelial cells. Electron microscopic evaluation of rat brains perfused with Tf-HRP at 4 degrees C and subsequently warmed to 37 degrees C for brief periods of time (2 minutes) showed sequestration of Tf-HRP in clathrin coated pits and vesicles on the luminal membrane of the brain endothelium. After 5 minutes of warming, diaminobenzidine (DAB) reaction product was present in vesicular structures 250-500 nm in diameter and in associated tubules morphologically identified as large or sorting endosomes. Recycling endosomes were also heavily labelled at this time point. Almost no DAB reaction product remained in the cerebral endothelial cells when the warming period was longer than 15 minutes. Other rat brains were perfused with Tf-HRP at 30 degrees C for 15 minutes prior to fixation and DAB cytochemistry. In these studies, brain endothelial cells contained large amounts of DAB reaction product, mostly localized in 50-100 nm vesicles and tubules, often in the Golgi region of the cells. Coated pits and vesicles and large endosomes were also heavily labelled. Transcytosis of Tf-HRP was not identified in either perfusion protocol. Ultrastructural, indirect immunocytochemical localization of transferrin receptors showed that the transferrin receptor is highly polarized at the blood-brain barrier and is localized only on the apical membrane, in contrast to other polarized epithelial cells, like hepatocytes, in which the receptor is present on the basolateral membrane. The evidence supports an iron transport model in which iron-loaded transferrin is taken up by receptor-mediated endocytosis at the luminal membrane of brain capillaries. The iron then dissociates from transferrin in endosomal compartments and is transcytosed by unknown mechanisms, while the transferrin is retroendocytosed.

Animals↗
PubMed1993

Apical-basal membrane polarity of membrane phosphatases in isolated capillary endothelium: alteration in ultrastructural localisation under culture conditions.

Capillaries from freshly isolated rat epididymal fat were subjected to protocols that allowed ultrastructural localisation of alkaline phosphatase and 5'-nucleotidase. Alkaline phosphatase was almost entirely restricted to the capillary luminal membrane and vesicles associated with this membrane. 5'-nucleotidase was localised on the basal or abluminal membrane and associated vesicles. Arterioles and occasional venules were also present in the cell isolates, and arteriole localisation of 5'-nucleotidase was identical to that in capillaries. In venules, 5'-nucleotidase often failed to exhibit a polarised distribution and was present on both membrane domains. In confluent cultured endothelial cells, 5'-nucleotidase was not expressed in a predominantly polarised arrangement. Alkaline phosphatase was found on apical surfaces and regions of lateral cell contact. The results of these studies show that capillary endothelial cells exhibit enzyme polarity of their surface membranes which is subject to change on introduction of the cells to tissue culture.

5'-Nucleotidase↗
PubMed1992

Studies of the mechanism of iron transport across the blood-brain barrier.

The mechanism by which iron enters the central nervous system from the blood is not well understood. Iron in blood plasma is totally bound to transferrin (Tf), a major plasma glycoprotein. Tf receptors are present on the blood-brain barrier (BBB) endothelium. It is not known whether iron separates from Tf during its passage across the endothelial cells and then enters the brain by another mechanism, or whether the two proteins enter the brain together. We characterize here the morphological pathway for endocytosis of a monomeric horseradish peroxidase-transferrin conjugate by the rat BBB endothelium. Our results indicate that this conjugate binds to Tf receptors on the luminal BBB, is internalized via clathrin-coated vesicles, enters early or sorting endosomes, and, subsequently, late or recycling endosomes near the Golgi apparatus. No evidence is found for Tf transcytosis. It is likely that iron separates from Tf in early endosomes, which are assumed to be acidic, as they are in other cells, and enters the brain by an as yet undefined pathway. A clonal line of brain capillary endothelial cells that mimics the BBB when grown on permeabilized membranes can transcytose iron provided as Fe55-Tf. This cell line may provide a useful system to determine the pathway that iron uses to enter the brain. We also present evidence that cultured chick embryo forebrain neurons contain a large number of a unique Tf receptor.

Animals↗
PubMed1992

Receptor-mediated endocytosis of insulin by cultured endothelial cells.

Label-fracture immunochemistry and pre-embedding indirect immunocytochemistry were applied to investigate insulin uptake by endothelial cells. Freeze fracture replicas showed that a small percentage of native insulin receptors are associated with non-coated pits (4%) and coated pits (2%). After warming, receptor bound insulin became increasingly associated with such endocytotic vesicles. After 2 min the percentage of detectable insulin associated with non-coated and coated pits increased to 16% and 8%, respectively. Pre-embedding immunocytochemical localization of insulin gave results consistent with those obtained from the label-fracture studies. Both non-coated and coated vesicles appeared labelled after 5 min of warming. Non-coated vesicles contained 25% of the cell associated insulin while 9% was associated with coated pits and vesicles. After 10 min of warming, 9% of label was located in non-coated vesicles and 7% in coated vesicles. A large proportion (29%) of the label was found in tubular-vesicular endosomes at this time. After 15 min of warming, 30% of the remaining cell-associated gold label was found in multivesicular bodies. These experiments demonstrate that insulin uptake by endothelium is mediated by both coated and non-coated vesicles and that, once internalized, insulin is routed through endosomal pathways that primarily result in transcytosis.

Animals↗
PubMed1992

Insulin-like growth factor binding protein (IGFBP)4 accounts for the connective tissue distribution of endothelial cell IGFBPs perfused through the isolated heart.

Insulin-like growth factor binding protein 4 (IGFBP4) was purified to homogeneity from conditioned media of bovine pulmonary artery endothelial cells and shown to have the N-terminal amino acid sequence DEAIHCPPCS, a sequence unique to IGFBP4. The IGFBP4 was separated into predominantly glycosylated and nonglycosylated fractions, with each fraction separately perfused through isolated, beating rat hearts. Both forms of IGFBP4 crossed the capillary boundary of the heart and distributed primarily in subendothelial connective tissue components with a connective tissue/cardiac muscle distribution ratio of 20:1 for the glycosylated fraction and 27:1 for the nonglycosylated fraction. Perfused IGFBP1, 2, 3, and IGF-I also crossed the capillary boundary but in contrast to IGFBP4, preferentially localized in cardiac muscle with a connective tissue/muscle ratio of approximately 1:3. We conclude that the connective tissue distribution previously reported for IGFBPs in conditioned media of pulmonary artery endothelial cells is due to IGFBP4.

Amino Acid Sequence↗
PubMed1991

Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages.

Mouse peritoneal macrophages were labeled with [1-3H]ethanolamine, and the presence of radioactive [3H]phosphatidylethanolamine (PE) at the plasma membrane was monitored by reacting the cells with trinitrobenzene sulfonic acid (TNBS) under nonpenetrating conditions. Macrophages stimulated with either the calcium ionophore A23187 or zymosan demonstrated a larger proportion of radiolabeled PE in the plasma membrane than control, nonstimulated cells. In experiments in which macrophages were labeled with ethanolamine for increasing times, appearance of membrane 3[H]PE was stimulated as early as after 2 hr of labeling. Macrophages labeled for 24 hr, then stimulated and returned to fresh medium still reflected a higher amount of membrane 3[H]PE at 2 hr after the stimulation, suggesting stimulation results in long-term alterations in plasma membrane lipids. Protease-peptone-elicited macrophages, which are not stimulated by zymosan or ionophore, did not exhibit an increase in membrane 3[H]PE upon stimulation. The size of the TNBS-accessible radiolabeled PE pool increased proportionately with a second stimulation; however, a subsequent labeling of the cells with TNBS after brief warming increased the TNBS-accessible pool in control cells only. As shown in previous studies, macrophage stimulation resulted in an increased incorporation of lipid precursors into phospholipid. The mass of plasma membrane Tnp-PE relative to mass of PE was not increased in ionophore-treated macrophages in contrast to a small (approximately 22%) increase in zymosan-treated cells. These results are suggestive of alterations in lipid synthesis in stimulated macrophages and possible long-term changes in the structure and function of the plasma membrane of macrophages following stimulation.

Animals↗
PubMed1991

Ultrastructural localization of arachidonic acid in stimulated macrophages.

The distribution of 3[H] arachidonic acid incorporated into cultured mouse peritoneal macrophages was assessed upon stimulation of the cells with either the calcium ionophore A23187 or zymosan. After a labeling time of 24 h, cells were stimulated and processed for light and electron microscopic autoradiography. Grains were primarily localized over the plasma membrane and lipid-containing vesicles of both control and stimulated cells. In macrophages stimulated with ionophore, a decreased labeling density was evident in both of these cell compartments. Similar alterations in labeling pattern were observed in zymosan treated cells, although a larger decline in grain density occurred from the plasma membrane compartment. Immunocytochemical localization of PGE2, a major eicosanoid product released upon ionophore stimulation, revealed the presence of the prostaglandin in clear vesicular structures, many of which appear to be continuous with the plasma membrane. These results provide morphological evidence that different cellular pools of arachidonic acid may be differentially mobilized for eicosanoid production as a function of the mode of stimulation.

Animals↗
PubMed1991

Plasma membrane phospholipid translocation in the mouse peritoneal macrophage: differential response to stimulation of eicosanoid production.

The metabolism and translocation of exogenously introduced plasma membrane phosphatidylcholine (PC) having the fluorescent fatty acid analog aminocaproyl NBD (N-nitrobenzo-2-oxa-1,3 diazole) (NBD-PC), in the sn2 position was studied in cultured murine peritoneal macrophages using biochemical and morphological techniques. Following labeling of the cell plasma membrane at 2 degrees C by vesicle lipid exchange, macrophages were warmed in the presence or absence of pharmacological stimuli of eicosanoid production and release. Fluorescence microscopy indicated that the phospholipid was translocated to an internal cellular pool upon stimulation with zymosan. In contrast, the membrane PC analog was primarily metabolized and released after being found diffusely associated with the cytoplasm in macrophages stimulated with the calcium ionophore A23187. Evidence obtained by double labeling zymosan-treated macrophages with NBD-PC and a monoclonal antibody directed against a lysosomal membrane protein demonstrated that the fluorescent lipid is internalized in association with the zymosan particles and both are found in lysosomes. The results suggest that multiple pathways exist in peritoneal macrophages which target plasma membrane PC into different cellular compartments for hydrolysis and conversion to eicosanoid products and release from cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Cytoskeletal organization and synthesis in substrate-independent and -dependent myogenesis in chick embryos.

Chick embryo myoblasts were fused in suspension culture to form myoballs by modification of previous procedures that excluded the use of divalent ion chelators and antimitotic drugs and included the continuous presence of serum in order to analyze the organized appearance and synthesis of major cytoskeletal proteins during cell attachment and spreading. The organization of the major cytoskeletal proteins actin, tubulin, and vimentin was assessed by fluorescence microscopy under these conditions as well as under conditions in which the myoballs were allowed to attach and spread on a collagen-coated substrate. Actin, detected by fluorescence microscopy, stained myoballs diffusely and was reorganized to form stress fibers in the attached and spreading myoball. Nuclei were segregated to a centrally located lattice of microtubules. The microtubule-specific drugs nocodazole and taxol prevented myoball spreading and the establishment of myotube polarity, respectively. Vimentin appeared as wavy ribbons in a perinuclear position around attached and spreading myoballs. In parallel studies, the synthesis of these cytoskeletal proteins was analyzed by radioisotopic labeling and polyacrylamide gel electrophoresis. These studies showed that myoballs possess altered ratios of actin and tubulin isoforms and of phosphorylated and nonphosphorylated vimentin compared to myotubes. These ratios rapidly change to the myotube pattern when myoballs are allowed to attach to solid substrata. Thus, although both myoballs and myotubes undergo muscle-specific differentiation, their cytoskeletal proteins are morphologically and biochemically distinct.

Actin Cytoskeleton↗