bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholesterol‐derivatized polyurethane: Characterization and endothelial cell adhesion
Authors: Stachelek Stanley J. , Alferiev Ivan , Choi Hoon , Kronsteiner Allyson , Uttayarat Pimporn , Gooch Keith J. , Composto Russell J. , Chen I‐Wei , Hebbel Robert P. , Levy Robert J.
Published: December 2004

Endothelialization of synthetic surfaces has been challenging with limited success thus far. We investigated the hypothesis that covalent attachment of cholesterol to polyurethane via the urethane nitrogen groups would create a high‐affinity surface for attachment and adhesion of endothelial cells. Cholesterol was covalently bound to the polyether polyurethane, Tecothane®, by first derivatizing the polyurethane nitrogen groups with bromoalkyl side chains, followed by reacting mercapto‐cholesterol to the bromoalkyl sites. Cholesterol‐modified polyurethane demonstrated a qualitatively smoother surface per atomic force microscopy than nonmodified and increased surface energy (contact angle measurements) compared with unmodified polyurethane. Cell attachment assays showed a significantly greater number of attached bovine arterial endothelial cells (p = 0.0003) after 45 min of seeding on cholesterol‐modified polyurethane versus unmodified polyurethane. Bovine arterial endothelial cells cultivated on cholesterol‐modified Tecothane® showed significantly greater levels of cell retention compared with unmodified Tecothane® when exposed to arterial level shear stress for 2 h (25 dynes/cm2) with 90.0 ± 6.23% cells remaining adherent compared with unmodified polyurethane, 41.4 ± 11.7%, p = 0.0070. Furthermore, ovine endothelial precursors, obtained as blood outgrowth endothelial cells, were seeded on cholesterol‐modified polyurethane and exposed to 25 dynes/cm2 shear conditions for 2 h, with the retention of 90.30 ± 3.25% of seeded cells versus unmodified polyurethane, which retained only 4.56 ± 0.85% (p < 0.001). It is concluded that covalently linking cholesterol to polyurethane results in improved material properties that permit increased endothelial cell retention compared with unmodified polyurethane. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 72A: 200–212, 2005
Effect of acidic pretreatment combined with a silane coupling agent on bonding durability to silicon oxide ceramic
Authors: Foxton Richard M. , Nakajima Masatoshi , Tagami Junji , Miura Hiroyuki
Published: December 2004

This study examined the effect of different acidic treatments and the role of a phosphate monomer in a silane coupling agent on the durability of the dual‐cure resin cement/silicon oxide bond. Ceramic blocks (Vita Celay Blanks) were cut into multiple 3 mm‐thick slices and polished using 600 grit SiC paper. Two pairs were left untreated [controls (CTRL)], two pairs were treated with 40% phosphoric acid and rinsed with water for 30 s (PA), and another two pairs treated with 20% hydrofluoric acid followed by 30 s water rinsing (HF). Half the specimens were silanated with Tokuso Ceramics Primer (TCP) (Tokuyama) and the other half with TCP formulated without phosphate monomer (TCP–NoPM). All the pairs were bonded with Bistite II dual‐cure resin cement (Tokuyama) and light cured. After 24 h water storage at 37°C, 0.7 mm‐thick slabs were serially sectioned. Immediately, after 6 months and after 1 year of water storage, two slabs were randomly selected from each subgroup, and sliced into beams (6 × 0.7 × 0.7 mm) for the microtensile bond strength (μTBS) test. The μTBS data were statistically analyzed using multiple Wilcoxon Signed Rank tests (p < 0.05). Failure modes were determined using a confocal laser‐scanning microscope. Ceramic surface morphology after the different acidic treatments was examined using an SEM. After 1 day, in the case of silane treatment with TCP, there were no significant differences in μTBS between the control and acid‐treated groups (p > 0.05), whereas with TCP–NoPM, the μTBS of the control was significantly lower than the acid‐treated groups (p < 0.05). All the TCP and acid‐treated TCP–NoPM groups exhibited significant reductions in μTBS after 6 months (p < 0.05). After 1 year, the μTBS of the acid‐treated TCP groups were not significantly different from the control TCP group (p > 0.05). There was also no significant difference between the HF‐treated TCP and TCP–NoPM groups (p > 0.05) after 1 year, all exhibiting greater than 10 MPa tensile bond strength. It is suggested that acidic pretreatment of the ceramic surface does not improve the durability of the dual‐cure resin cement/silicon oxide ceramic bond when an acidic phosphate monomer is present as an activator in a ceramic primer. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 73B: 97–103, 2005
High α‐amylase activity in the syncytiotrophoblastic cells of first‐trimester human placentas
Authors: Fisher Susan J. , Laine Roger A.
Published: December 2004

The syncytiotrophoblastic brush border of the human placenta forms the maternal‐fetal interface and is an important determinant of placental function. Electron micrographs of fresh brush border preparations isolated from first‐trimester human placentas showed membrane vesicles, open‐ended microvilli, and numerous glycogen particles. Analysis of the microvillar membranes for several plasma and intracellular membrane markers showed a high degree of purification, comparable to the results reported for the isolation of microvilli from full‐term human placentas. The microvillar preparations from first‐trimester placentas, however, also contained the enzymes necessary to synthesize and degrade glycogen. The degradation resulted in the accumulation of maltotriose and maltotetraose, apparently due to the action of a liver‐type α‐amylase. The occurrence of this enzyme and the enzymes for synthesizing glycogen in this brush border fraction is probably associated with the necessity for an extremely active glucose transport and liver‐like storage system within the fetal tissue at this fetal‐maternal membrane interface.
Epidermal growth factor receptors on PC12 cells: Alteration of binding properties by lectins
Authors: Vale Ronald D. , Shooter Eric M.
Published: December 2004

The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of 125I‐EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37°C and 4°C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylalion of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with 125I‐NGF binding, WGA but not Con A was found to increase, by scveralfold; the proportion of 125I‐EGF binding that is resistant to solubilization by Triton X‐100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.
Mitochondrial DNA of an African trypanosome
Authors: Stuart Kenneth
Published: December 2004

The maxicircles of African trypanosome kDNA are the genetic equivalent of other mitochondria! DNAs, but the function of minicircles is unknown. The maxicircle of Trypanosoma brucei 164 encodes conventional mitochondrial gene products and is largely but not completely transcribed. Nucleotide sequence analysis of a region not found to be transcribed revealed numerous translation termination codons in all three reading frames of both strands and numerous inverted repeats, suggesting that this segment does not have polypeptide‐coding function. This segment may encode a t‐RNA and has a sequence resembling a consensus sequence found in mitochondrial introns, thus implying that transcript processing occurs in trypanosome mitochondria. While several cloned minicircles had distinct restriction maps reflecting T brucei minicircle heterogeneity, one segment of the minicircle contained a sequence that was conserved by minicirclcs from other trypanosome strains and species. Of nine mutants unable to grow as the respiring procyclic forms, seven were devoid of kDNA. The other two mutants retained normal amounts of all maxicircle restriction fragments and normal amounts of those minicircle sequences tested. Minicircle alterations probably occur in these mutants, since the kDNA docs not stain with Giemsa and bands at an altered density in cesium chloride/ethidium bromide density gradients.
Killing of Giardia lamblia trophozoites by normal human milk
Authors: Gillin Frances D. , Reiner David S. , Wang Chi‐Sun
Published: December 2004

The clinical course of giardiasis is variable, and serum antibodies do not appear to be protective. We propose that natural factors either produced by intestinal tissue, transported into the intestine, or ingested (ie, by breast‐fed babies) might promote resistance to this disease. Human milk is very rich in secretory IgA (S‐IgA) antibodies, as well as nonspecific antibacterial factors (eg, lactoferrin, lysozyme).

Previous studies showed that Giardia lamblia trophozoites were killed by nonimmune human milk (NHM) in a time‐ and concentration‐dependent manner. Removal of >99% of the S‐IgA from NHM did not decrease its Giardia‐cidal activity. Thus, the killing was not antibody dependent. This is the first demonstration of nonimmune antiparasitic defenses in human milk.

The present studies show that in the presence of NHM, trophozoites lost motility, swelled, and lysed. The Giardia‐cidal activity (GCA) may be specific to human milk, since unhcated cow's and goat's milk were virtually devoid of activity. Much, but not all, of the GCA was lost when NHM was heated or reacted with diisopropylfluorophosphate (DIFP), a specific esterase inhibitor. Activity of the major human milk lipase (BSL, bile salt‐stimulated lipase, a fatty acid esterase) was lost after heat or DIFP treatment and was absent from cow's or goat's milk. The parasites were also killed by pure BSL. These studies suggest that BSL may be a heat‐labile Giardia‐cidal component of NHM.
Interaction of high‐density lipoprotein with Trypanosoma brucei: Effect of membrane stabilizers
Authors: Rifkin M. R.
Published: December 2004

The specific lysis of bloodstream trypanosomes by serum from a nonpermissive mammalian host is the result of interaction between the serum trypanocidal factor (high‐density lipoprotein) and the trypanosome surface. The studies described in this paper attempt to define further the mode of action of this cytotoxic lipoprotein. The binding of high‐density lipoprotein to Trypanosoma brucei was instantaneous at 4°C and readily reversible. Binding was not mediated by the surface glycoprotein as removal of the surface coat enhanced binding at 4°C, and no stable glycoprotein‐lipoprotein complex could be detected. Pretreatment of trypanosomes with the cross linker dimethylsuberimidate rendered cells resistant to lysis. Addition of membrane‐stabilizing drugs, such as cytochalasins C, D, and E, and local anesthetics (dibucaine, tetracaine, and procaine), also inhibited high‐density lipoprotein‐induced cell lysis. The data presented support the idea that at 37°C lateral diffusion of the variant surface glycoprotein, an integral membrane protein, allows maximal high‐density lipoprotein‐cell interaction in serum‐sensitive cells, and that altered properties of the plasma membrane induced by low temperature or the addition of cytochalasins, local anesthetics, or zinc inhibit this interaction, possibly by increasing the shielding of the plasma membrane by more rigidly anchored surface glycoprotein molecules.
The light‐dependent control of chloroplast development in barley (Hordeum vulgare L)
Authors: Apel Klaus , Gollmer Ingrid , Batschauer Alfred
Published: December 2004

The light‐induced greening of etiolated barley plants is used as a model to study the light‐dependent control of plastid development. Upon illumination a rapid transformation of etioplasts to chloroplasts is induced. The effect of illumination docs not only include the light‐dependent chlorophyll synthesis but also the appearance or decline of specific proteins within the plastid membrane fractions. So far two of these proteins have been studied in detail. The light‐harvesting chlorophyll a/b protein (LHCP) is one of the major protein constituents of the thylakoid membrane of chloroplasts. However, this protein is not detectable among the membrane polypeptides of etioplasts. Illumination of dark‐grown bar‐Icy plants induces a massive insertion of the LHCP, The appearance of the protein is controlled by the cooperation of at least two distinct photoreceptors: protochlorophyllide and phytochrome. In dark‐grown barley plants not only the LHCP but also its mRNA is not detectable. The light‐dependent appearance of mRNA activity for the LHCP is under the control of phytochrome (Pfr). Even though the appearance of mRNA activity is induced via Pfr by a single red light pulse, the assembly of the complete LHCP takes place only under continuous illumination, which allows chlorophyll synthesis. The second protein analyzed so far is the NADPH‐prolochlorophyllide‐oxidoreductase. This enzyme catalyzes the light‐dependent reduction of protochlorophyllide to chlorophyllide and thus controls one of the first detectable light‐dependent reactions during the greening period. It is generally assumed that this enzyme is responsible for the overall chlorophyll synthesis and accumulation during the greening period. In contrast to this hypothesis, we found a rapid decline of the enzyme during illumination. In addition to the decrease of the enzyme protein, the translatable mRNA coding for the enzyme also declines rapidly under the influence of light. Also this effect is mediated by phytochrome. Using cloned cDNA as hybridization probes we have demonstrated that the light‐induced changes of the two translatable mRNAs for the NADPH‐protochlorophyllide oxidoreductase and the LHCP are both paralleled by corresponding changes in the steady‐state concentration of the mRNA sequences. Thus, it seems likely that one major point of control at which phytochrome regulates the development of chloroplasts is the expression of genes at the level of transcription.
Substrate and phospholipid specificity of the purified mannitol permease of Escherichia coli
Authors: Jacobson Gary R. , Tanney Laura E. , Kelly Dori M. , Palman Karin B. , Corn Stephen B.
Published: December 2004

D‐Mannitol is transported and phosphorylated by a specific enzyme II of the phosphotransferase system of Escherichia coli. This protein was purified previously in detergent solution and has been partially characterized. As one approach in understanding the structure and mechanism of this enzyme/pcrmease, we have tested a number of sugar alcohols and their derivatives as substrates and/or inhibitors of this protein. Our results show that the mannitol permease is highly, but not absolutely, specific for D‐mannitol. Compounds accepted by the enzyme include those with substitutions in the C‐2 (= C‐5) position of the carbon backbone of the natural substrate as well as D‐mannonic acid, one heptilol and one pentitol. All of these compounds were both inhibitors and substrates for the mannitol permease except for D‐mannoheptilol, which was an inhibitor but was not phosphorylated by the enzyme. No compound examined, however, exhibited an affinity for the enzyme as high as that for its natural substrate. We have also investigated the phospholipid requirements of the mannitol permease using phosphotipids purified from E coli. The purified protein was significantly activated by phosphatidylethanolamine, but little activation was observed with phosphatidylglycerol or cardioli‐pin. These observations partially delineate requirements for interaction of sugar alcohols and phospholipids with the mannitol permease. They suggest approaches for the design of specific active site probes for the protein, and strategies for stabilizing the enzyme's activity in vitro.
Granulocyte macrophage‐colony stimulating factor stimulates the synthesis of membrane and nuclear proteins in murine neutrophils
Authors: Stanley I. J. , Burgess A. W.
Published: December 2004

The effect of granulocyte macrophage‐colony stimulating factor (GM‐CSF), a well‐characterized hemopoietic regulator, on protein synthesis in murine bone marrow neutrophils is described. Bone marrow neutrophils in excess of 95% purity were obtained by fluorescence‐activated cell sorting.While GM‐CSF did not appear to slow the rate of dying of peritoneal exudate neutrophils or thymus cells, the viability of bone marrow neutrophils after 17 hr was enhanced (40%) by GM‐CSF. GM‐CFS had no effect on total 35S‐methionine incorporation by thymocytcs or peritoneal exudate neutrophils over a 17‐hr incubation period; however, bone marrow neutrophils showed increased incorporation (approximately 10%) at all times between 5–17 hr. As viability and 35S‐methionine incorporation of bone marrow neutrophils at 5 hr,were minimally affected by GM‐CSF, this time point was chosen to study the effect of GM‐CSF on the synthesis of particular proteins. Two‐dimensional polyacrylamide gels of 35S‐methionine‐labelled lysates were prepared from whole cells, isolated nuclei, and membranes. Quantitative analysis of the fluorograms obtained from the two‐dimensional electropherograms by a computer‐linked optical data digitiser indicated that out of a total of 180 proteins, the amount of label contained in 11 proteins was significantly higher in the presence of GM‐CSF, while.three proteins, apparently of cytoplasmic origin, contained less label than control cells. Eight of these proteins were identified as nuclear, and one was membrane derived. Attempts have been made to identify some of the inducible proteins and to correlate results with other studies of normal hemopoietic and Icukcmic cells. The significance and multiple functions of GM‐CSF in hemopoiesis are discussed.
The ubiquitin‐mediated proteolytic pathway and mechanisms of energy‐dependent intracellular protein degradation
Authors: Ciechanover Aaron , Finley Daniel , Varshavsky Alexander
Published: December 2004

In this review we briefly describe the lysosomal system, consider the evidence for multiplicity of protein degradation pathways in vivo, discuss in detail the ubiquitin‐mediated pathway of intracellular ATP‐dependent protein degradation, and also the possible significance of ubiquitin‐histone conjugates in chromatin. For detailed discussions of the various characteristics and physiological roles of intracellular protein breakdown, the reader is referred to earlier reviews [1–7] and reports of recent symposia [8–10]. Information on the ubiquitin system prior to 1981 was described in an earlier review [11]. Hershko has briefly reviewed more recent information [12].
Hepatocyte receptors for antithrombin III‐proteinase complexes
Authors: Fuchs Herbert E. , Shifman Mark A. , Michalopoulos George , Pizzo Salvatore V.
Published: December 2004

The in vivo clearance of antithrombin III‐proteinase complexes occurs via a specific and saturable pathway located on hepatocytes. We now report studies of the catabolism of antithrombin III‐proteinase complexes in vitro using rat hepatocytes in primary culture. Antithrombin III‐thrombin and trypsin complexes were prepared and purified to homogeneity. Ligand uptake by hepatocytes was concentration, temperature, and time dependent. Initial rate studies were performed to characterize the maximum rate of uptake, V, and apparent Michaelis constant Kapp. These studies yielded a V of 12.8 fmol/mg cell protein/min and a Kapp of 144 nM for antithrombin‐trypsin complexes. Competition experiments with antithrombin III, antithrombin III‐proteinase complexes, α2‐macroglobulin‐methylamine, asialoorosomucoid and the neoglycoproteins, fucosyl‐bovine serum albumin (BSA), N‐acetylglucosammyl‐BSA, and mannosyl‐BSA indicated that only antithrombin III‐proteinase complexes were recognized by the hepatocyte receptor. Uptake studies were performed at 37°C with 125I‐antithrombin III‐trypsin and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in conjunction with autoradiography. These studies demonstrate time‐dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time‐dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions.
Secretion and degradation of mutant leucine‐specific binding protein molecules containing C‐terminal deletions
Authors: Landick Robert , Duncan James R. , Copeland Bruce R. , Nazos Penelope M. , Oxender Dale L.
Published: December 2004

The leucine‐specific binding protein (LS‐BP), a periplasmic component of the Escherichia coli high‐affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N‐terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS‐BP gene have been constructed with altered or missing amino acid sequences in the C‐terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.
Characterization of a 140Kd cell surface glycoprotein involved in myoblast adhesion
Authors: Chapman Ann E.
Published: December 2004

Two monoclonal antibodies that cause changes in the morphology of cultured chick myogenic cells have been described previously [8]. In this paper, these antibodies are shown to interact with the same 140Kd protein. The 140Kd protein has been further characterized as a cell‐surface glycoprotein by lactoperoxidase‐catalyzed iodinations and lectin affinity chromatography. The protein is resistant to digestion by trypsin and collagenase and has been shown to be unrelated to fibronectin by immunoprecipitation studies and by peptide mapping. A second protein, of approximately 170Kd MW; is also immunoprecipitated by the monoclonal antibodies. This protein is probably unrelated to the 140Kd protein since the peptide maps are quite distinct.
Replacement perfusion of cultured eucaryotic cells: A method for the accurate measurement of the rates of growth, protein synthesis, and protein turnover
Authors: Spanier Arthur M. , Clark William A. , Zak Radovan
Published: December 2004

The fractional rates of protein synthesis (ks) and degradation (kp) were studied in the myeloma cell line SP2/0‐AG14 grown at different rates (kg). Cells in spinner flask suspension cultures were maintained at constant cellular density for prolonged periods by replacement perfusion of labeling medium at a rate equivalent to the rate of growth. Total protein synthesis was calculated from the specific‐radioactivity of labeled L‐leucine in the precursor (medium) and cellular protein. Fractional synthesis rates determined by approach to equilibrium labeling were the same as those determined by equilibrium‐pulse labeling kinetics and pulse‐chase kinetics. The rate of protein degradation was determined from the established relationship kg = ks – kp. Protein synthesis rates remained constant over a threefold range in the rate of cell growth. At relatively slow growth rates (kg = 0.017/hr) turnover represented a major fraction of total synthesis (kp = 0.032/hr = 0.65ks). At rapid growth rates (kg = 0.058/hr) the value of kp was less than 0.005/hr. No major difference was observed between the ks determined for individual cellular proteins (separated by SDS‐polyacrylamide (7.5%) gel electro‐phoresis) from rapid‐ and slow‐growing cultures. Thus, with an invariable ks, any change in growth rate is due to an inverse change in the rate of turnover. Since turnover is the balance between synthesis and degradation and since synthesis is unchanging then changes in the growth rate of SP2/0‐AG14 should be due to changes in the rate of protein degradation. Experiments were therefore performed to determine the origin of the degradative machinery, ie, cytosolic or lysosomal; autolysis of prelabeled cellular protein (in vitro) was observed only at acidic pH (4.2) and WUS totally inhibited by addition of lcupcptin (10 μM) and pepstatin (2 μM), the specific inhibitors of lysosomal cathepsins B (L) and D, respectively. Since growth rate appears to be regulated by the alterations in the rate of protein degradation and degradation (in vitro) in SP2/0‐AG14 appearsto be lysosomal, then one should be able to alter the rate of cellular growth by interfering with rate of lysosomal proteolysis. Indeed, when the lysosomotropic amine NH 4Cl (10 mM) is added to cells growing with a kg of 0.018/hr ± 0.001 (ks = 0.050/hr ± 0.002) the growth rate increased to 0.051/hr ± 0.002 without change in the rate of protein synthesis (ks = 0.049/hr ± 0.003). It is suggested from our data that the cellular growth rate of SP2/0‐AG14 is regulated by the lysosomal apparatus; whether this regulation is itself regulated by either a specific compartmentalization of the lysosomal proteinases and/or their substrates or by endogenous protease inhibitors, should prove to be an exciting area for future investigation.
Concepts of epithelial‐mesenchymal interactions during development: Tooth and lung organogenesis
Authors: Slavkin Harold C. , Snead Malcolm L. , Zeichner‐David Margarita , Jaskoll Tina F. , Smith Barry T.
Published: December 2004

One of the major problems in developmental biology concerns how differential gene activity is regionally controlled. One approach to this problem is the use of mesenchyme specification of epithelial‐specific gene expression, such as, during tooth morphogenesis or lung morphogenesis. In the example of tooth morphogenesis, dental papilla ectomcsenchyme induces de novo gene expression as assayed by detection of amelogenin transcripts, or immunodetection of amelogenin poly‐peptidcs within ameloblast cells. This process does not require serum supplementation or exogenous factors during epithelial‐mesenchymal interactions in vitro. In contrast, lung morphogenesis requires hormones to mediate mesenchyme‐derived influences upon type II epithelial cell differentiation and the production of pulmonary surfactant (eg, neutral and phospholipids, surfactant proteins). Glucocorticoids are required to stimulate the release of fetal pneumonocyte factor (FPF) from fibroblasts which, in turn, enhance the production of pulmonary surfactant. Thy‐roxin appears to regulate the relative responsiveness of progenitor type II cells to steroid‐stimulated release of FPF. This review will highlight key concepts associated with these developing organ systems and emphasize the problem of regional controls which regulate epithelial cell‐specific gene activity.
Degradation of extracellular matrix by the trophoblastic cells of first‐trimester human placentas
Authors: Fisher Susan J. , Leitch Mark S. , Kantor Marsha S. , Basbaum Carol B. , Kramer Randall H.
Published: December 2004

First‐trimester human placental villi were cultured on 3H‐leucine‐labeled extracellular matrices isolated from the PF HR9 and PYS‐2 cell lines. Both cell lines produced an extracellular matrix that contained basement membrane‐specific macromolecules, including type IV collagen, laminin and proteoglycan. Both matrices promoted outgrowth of cells from the villi which, according to morphological criteria, were identified as cytotrophoblastic cells. As the cells migrated from the attachment site, they caused a marked focal dissolution of the matrix which was accompanied by a concomitant release of 3H‐labeled material into the media. Approximately half of this material chromatographed near the inclusion volume of Sephadex G‐50, indicating that the labeled matrix components had been degraded. This phenomenon was dependent on the age of the placenta. Second‐trimester placental villi also adhered to the matrix, but no areas of dissolution were formed and no significant amounts of radioactivity were released into the medium. These results suggest that culture of first‐trimester human placental villi on extracellular matrices may be useful for the study of some of the early embryonic events leading to human implantation, during which the trophoblastic cells erode the uterine epithelium.
Matrix‐cytoskeletal interactions in the developing eye
Authors: Hay Elizabeth D.
Published: December 2004

The embryonic avian corneal epithelium in vitro responds to extracellular matrix (ECM) molecules in either soluble or polymerized form by flattening its basal surface, organizing the basal cortical actin cytoskeleton, and stepping up its production of corneal stroma twofold. Embryonic corncal epithelia, like hepatocytes and mammary gland cells, seem to contain heparan sulfate proieoglycan (HSPG) in their plasmalemma, which may interact with actin on the one hand or underlying collagen on the other. Work on the corneal epithelium suggests that, in addition to HSPG, specific glycoprotein receptors for laminin and collagen exist in the basal plasmalemma and play, the critical role in actually organizing the basal epithelial cytoskeleton. As yet. uncharacterized proteins may link such receptors to actin. We suggest that ECM‐dependent organization of the cytoskeleton is responsible for ECM enhancement of corneal epithelial differentiation. Cell shape and exogenous ECM also affect mesenchymal cell differentiation. In the case of the conical fibroblast migrating in collagen gels, an actin cortex present around the elongate cell seems to interact with myosin in the cytosol to bring about pseudopodial extension. Both microtubules and actin microfilaments are involved in fibroblast elongation in collagen gels. It follows from the rules presented in this review that the mesenchymal cell surface is quite different from the epithelial cell surface in its organization. Nevertheless, epithelial cell surface‐ECM interaction can be modified in the embryo at particular times to permit predesignated epithelial‐mesenchymal transformations, as for example at the primitive streak. Though basal surfaces of definitive, nonmalignanl epithelia adhere rather strictly to the rules of epithelium‐ECM interaction and do not invade underlying ECM, the environment can be manipulated in vitro to cause these epithelia to send out pseudopodia and give rise aberrantly to mesenchymal cells in collagen gels. Further study of this phenomenon should cast light on the manner in which epithelial and mesenchymal cells organize receptors for matrix molecules on their cell surfaces and develop appropriate cytoskeletal responses to the extracellular matrix.
Liver tyrosine kinase activation during early stages of chemical hepatocarcinogenesis
Authors: Olson Jack W.
Published: December 2004

Protein phosphorylation at tyrosine residues is believed to he involved in several important cellular processes because tyrosine‐specific protein kinase activation is associated with stimulation of cellular proliferation by hormones and growth factors, embryogenesis, and retroviral cell transformation. Because cell proliferation is thought to be an essential component of chemical carcinogenesis. liver tyrosine‐specific protein kinase activity was examined during the early stages of the Solt and Farber chemical hepatocarcinogenesis model. Rats were given diethylnitrosamine in one dose (200 mg/kg, IP) followed by 2 weeks of dietary 0.02% 2‐acctylamino‐fluorene starting at day 14 after diethylnitrosamine, followed by partial hepatectomy on day 21. By day 32 this regimen produces a relatively synchronized population of hyperplastic liver nodules up to 1.5 mm in diameter. Rats were sacrificed on day 32, their livers were perfused with cold normal saline, homogenized, and centrifuged at 1,000g for 10 min. The resulting supernatant was centrifuged at 30,000g for 30 min and the pellet was assayed for tyrosine kinase activity using the synthetic peptide [Val5]angiotensin II as substrate. Rats that received the complete regimen had a 2.6‐fold increase in their liver tyrosine kinase activity as compared to sham controls (2.4 pmoles/min/mg protein vs 6.4 pmoles/min/mg protein, P < .05). In contrast, rats that received a partial regimen (ie, partial hepatectomy, or 2‐acetylaminofluorene + partial hepatectomy, or diethylnitrosamine + 2‐acetylaminofluorene) did not have elevated tyrosine kinase activity nor did they have hyperplastic nodules. These preliminary data suggest that activation of liver tyrosine kinase is associated with the very early stages of chemical hepatocarcinogenesis.
How do the migratory and adhesive properties of the neural crest govern ganglia formation in the avian peripheral nervous system?
Authors: Duband Jean Loup , Tucker Gordon C. , Poole Thomas J. , Vincent Michel , Aoyama Hirohiko , Thiery Jean Paul
Published: December 2004

The peripheral nervous system derives mainly from the neural crest both in the head and trunk. Using markers such as fibronectin (FN), neural cell‐adhesion molecule (NCAM), the nuclcolar marker for quail cells in chimaeric embryos, and NC‐1, a monoclonal antibody specific to crest cells and their neural derivatives, we have attempted to reconstruct the processes that lead to the formation of peripheral ganglia. Our observations allow us to propose a model of the formation of ganglia based on morphogenetic movements and on variations of crest cell adhesiveness. In most cases, crest cells migrate in morphologically defined and transient pathways that lead them to their final site of arrest; these pathways are always associated with FN, which appears necessary for crest cell attachment and movement in vitro. The directionality of crest cell migration is probably dictated by the cells' motile properties and population pressure in restricted areas suitable for cell movement. The disappearance of the pathways and of the substrate necessary for migration while the population is rapidly dividing may be responsible for the aggregation of crest cells in the case of the sensory ganglia. To the contrary, the aggregation of crest cells into autonomic ganglia (sympathetic, enteric, aid ciliary ganglia) does not seem to obey the same rules, no disappearance of the substratum or of the pathways being obvious; rather, their formation seems correlated with the de novo synthesis of adhesive molecules such as NCAM.
Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes
Authors: Vertel Barbara M. , Barkman Linda L. , Morrell Jeffrey J.
Published: December 2004

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5‐D‐4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5‐D‐4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5‐D‐4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose‐rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannos oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N‐acetyl‐D‐glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5‐D‐4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti‐CSPG monomer and not with anti‐type II collagen contain precursors of CSPG monomer not yet modified by Golgi‐mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti‐keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.
Further biochemical and physicochemical characterization of minor disulfide‐bonded (type IX) collagen, extracted from foetal calf cartilage
Authors: Ricard‐Blum Sylvie , Tiollier Jérôme , Garrone Robert , Herbage Daniel
Published: December 2004

Minor disulfide‐bonded collagen (previously termed X1–X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule, X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin‐sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80–100 nm and 40–55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30°C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin‐sensitive sites.
Formyl peptide chemotaxis receptors on the rat neutrophil: Experimental evidence for negative cooperativity
Authors: Marasco Wayne A. , Feltner Douglas E. , Ward Peter A.
Published: December 2004

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady‐state binding of f Met‐Leu‐[3H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analyses of the binding data indicated that a two‐saturable‐sites model was preferable to a one‐saturable‐site model and statistically valid by the F‐test (P < 0.1). In addition, Hill coefficients of 0.80 ± 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site‐site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met‐Leu‐Phe accelerated the dissociation of f Met‐Leu‐[3H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met‐Leu‐[3H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met‐Leu‐Phe used in the dilution medium; at the highest concentration tested (10,000‐fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone, In addition, occupancy‐dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor‐bound labeled ligand. These data showed that the f Met‐Leu‐[3H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met‐Leu‐[3H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met‐Leu‐Phe remained occupancy‐dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.
In situ hybridization of putative somatostatin mRNA within hypothalamus of the rat using synthetic oligonucleotide probes
Authors: Arentzen Rene , Baldino Frank , Davis Leonard G. , Higgins Gerald A. , Lin Yuan , Manning Robert W. , Wolfson Betty
Published: December 2004

The distribution of mRNA with high sequence homology to somatostatin mRNA within the periventricular hypothalamus of rat was assessed using in situ hybridization techniques with synthetic oligodeoxyribonucleotide probes, complementary to the 3′ coding region of rat somatostatin mRNA. The probes (22‐ and 24‐mers) were 5′‐end labeled using T4 polynucleotide kinase and γ‐32P‐ATP. They were used either individually or after ligation with T4 DNA ligase to form a 46‐mer. Serial tissue sections ( < 10 μm) were taken from the level of the preoptic/anterior hypothalamus through the paraventricular hypothalamus. In situ hybridizations were conducted at room temperature in hybridization buffer. Neurons immuno‐reactive with antiserum raised against somatostatin were identified in alternate sections using standard immunocytochemical procedures. The anatomical location of the hybridization signal was determined by autoradiography. Our results show that the peri‐ and paraventricular hypothalamus is rich in transcripts putatively coding for somatostatin and that these transcripts are co‐distributed with neurons immunoreactive with antisomatostatin immunoglobulin.
A β‐type transforming growth factor, present in conditioned cell culture medium independent of cell transformation, may derive from serum
Authors: Stromberg Kurt , Twardzik Daniel R.
Published: December 2004

An alpha‐type transforming growth factor (TGFα) is produced at high levels by rat embryo cells transformed by the Snyder‐Theilen strain of feline sarcoma virus (FeSV). Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF‐dependent growth factor of the TGF beta type (TGFβ) in this conditioned medium. This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography. This extracellular type of TGFβ activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation. Moreover, the presence of an EGF‐dependent. 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests scrum as an origin for the B‐type transforming growth factor initially observed in conditioned medium of Snyder‐Theilen FeSV transformed cells. This does not, however, preclude the possibility that TGFβ is also secreted by the transformed rat embryo cells themselves.
Stimulation of phospholipase A2 activity by oxygen‐derived free radicals in isolated brain capillaries
Authors: Au A. M. , Chan P. H. , Fishman R. A.
Published: December 2004

An exogenous free radical generating system added to isolated brain capillaries induces degradation of phospholipids. This inductive effect reflects increased phospholipase activities as measured by fatty acid composition of various phospholipid fractions. The correlation of phospholipid degradation with stimulation of phospholipases was further investigated by using cationic amphiphilic agents, which are known to be phospholipase A2 inhibitors. The breakdown of phospholipids was inhibited by the pretreatment of isolated capillaries with these drugs.
Biosynthesis of collagen
Authors: Fessler John H. , Doege Kurt J. , Duncan Keith G. , Fessler Liselotte I.
Published: December 2004

During the biosynthesis and assembly of collagen structures, disulfide links can serve several functions. During biosynthesis they successively stabilize intra‐peptide folding and associations of three chains into one molecule. Studies on the refolding and reassociation of reduced and denatured carboxyl propeptides of procollagen I showed that successive interactions of folding and assembly are successively weaker. Disulfide bridges were reestablished within correctly refolded carboxyl propeptides. Rearrangements of disulfide bridges may occur during the processing of type V procollagen molecules as these collagens become incorporated into extracellular matrix. The basement membrane procollagen IV molecules become disulfide linked at each end into networks, and there are indications that further rearrangements of disulfide links may allow additional modulation.
Recent advances in research on fibronectin and other cell attachment proteins
Authors: Yamada Kenneth M. , Akiyama Steven K. , Hasegawa Takayuki , Hasegawa Etsuko , Humphries Martin J. , Kennedy Dorothy W. , Nagata Kazuhiro , Urushihara Hideko , Olden Kenneth , Chen Wen‐Tien
Published: December 2004

Fibronectin and other cell attachment proteins provide molecular models for beginning to unravel the complex interactions of the cell surface with the extracellular matrix. This area has been reviewed in considerable detail previously [1–10]. Our brief review will therefore be selective rather than comprehensive, and it will focus on some recent generalizations about this class of proteins, as well as on recent advances in the molecular analysis of the functions of these proteins and their receptors. we shall also present various popular or provocative hypotheses and speculations about future work in the field.
Phosphorylation of the insulin receptor by casein kinase I
Authors: Tuazon Polygena T. , Pang Dennis T. , Shafer Jules A. , Traugh Jolinda A.
Published: December 2004

Insulin receptor was examined as a substrate for the multipotential protein kinasc casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the α2β2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the α and β subunits. The majority of the phosphate was associated with the β subunit with minor phosphorylation of the α subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated α2β2 receptor purified by affinity chromatography on immobilized O‐phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated α2β2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the β subunit.