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At least 1,729 records · Page 96Linked to original sources
PubMed2001

Monitoring signal transduction in cancer: from chips to fish.

The microarray format of RNA transcript analysis should provide new clues to carcinogenic processes. Because of the complex and heterogeneous nature of most tumor samples, histochemical techniques, particularly RNA fluorescent in situ hybridization (FISH), are required to test the predictions from microarray expression experiments. Here we describe our approach to verify new microarray data by examining RNA expression levels of five to seven different transcripts in a very few cells via FISH. (J Histochem Cytochem 49:925-926, 2001)

Biomarkers, Tumor↗
PubMedDate not supplied

The stability of amikacin, gentamicin, and tobramycin in total nutrient admixtures.

Amikacin (A), gentamicin (G), and tobramycin (T) were added to eight different total nutrient admixtures (TNA) with varying concentrations of dextrose, amino acid, and fat emulsion to determine drug and emulsion stability. All TNA were prepared aseptically and stored at room temperature under normal room lighting for 12 hr before drug addition. One volume of each drug was added to an equal volume of each of the eight TNAs to simulate 1:1 piggyback contact volumes. Samples were left at room temperature for 6 hr. Drug concentrations were analyzed by fluorescence polarization immunoassay. TNA/drug admixtures were pH tested and visually inspected before and after centrifugation in microhematocrit tubes, noting signs of emulsion stability at 1 and 6 hr. Emulsion particle size was determined at 1 and 6 hr using interference contrast microscopy. All three drugs retained their immunoreactivity in all TNAs for at least 6 hr. G and T were stable in all eight TNAs for at least 6 hr with no significant effect on emulsion particle size or stability after centrifugation. A was incompatible with all eight TNAs, resulting in visual breaking of all emulsions within 1 hr. Therefore, G and T, but not A, can be administered via piggyback method with the eight TNAs tested if the infusion is completed within 6 hr.

Amikacin↗
PubMed1990

Improved method for obtaining 3-microns cryosections for immunocytochemistry.

The following describes a modified technique for obtaining 3-microns sections for light microscopic level immunocytochemistry. By combining 20% sucrose with Tissue-Tek OCT embedding compound in a ratio of 2:1, we produced a block that was suitable for cutting 3-microns sections on a conventional cryostat. The 3-microns sections were dramatically improved compared with 10-microns sections cut from tissue embedded in OCT alone, when viewed with both differential interference contrast microscopy (Nomarski optics) and indirect immunofluorescence. The method is simple, uses materials already available, and does not require training in a new technique.

Cryopreservation↗
PubMed2003

The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion.

Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collagen-induced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VInull platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VInull or FcR-gammanull animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VInull platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils.

Animals↗
PubMed2006

Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3.

Mature megakaryocytes form structures called proplatelets that serve as conduits for platelet packaging and release at vascular sinusoids. Since the megakaryocyte expresses abundant levels of integrin alpha IIb beta3, we have examined a role for fibrinogen in proplatelet development and platelet release alongside that of other matrices. Primary mature murine megakaryocytes from bone marrow aspirates readily formed proplatelets when plated on fibrinogen at a degree that was significantly higher than that seen on other matrices. In addition, alpha IIb beta3 was essential for proplatelet formation on fibrinogen, as megakaryocytes failed to develop proplatelets in the presence of alpha IIb beta3 antagonists. Interestingly, inhibition of Src kinases or Ca2+ release did not inhibit proplatelet formation, indicating that alpha IIb beta3-mediated outside-in signals are not required for this response. Immunohistochemical studies demonstrated that fibrinogen is localized to the bone marrow sinusoids, a location that would allow it to readily influence platelet release. Further, thrombopoietin-stimulated alpha IIb-/- mice had a reduced increase in platelet number relative to controls. A similar observation was not observed for platelet recovery in alpha IIb-/- mice in response to antibody-induced thrombocytopenia, indicating the existence of additional pathways of regulation of proplatelet formation. These results demonstrate that fibrinogen is able to regulate proplatelet formation via integrin alpha IIb beta3.

Animals↗
PubMed2000

Aerosol delivery of muramyl dipeptide to rodent lungs.

Tuberculosis is the single most serious infectious disease worldwide. The respiratory tract is the primary site of infection by Mycobacterium tuberculosis(MTB). A number of immunogenic components of the cell wall of MTB, if delivered to the lungs as aerosols, can be used to study the local immune response. The site of deposition of these aerosols can be employed to control their residence time in the lungs. Muramyl dipeptide (MDP) aerosols were delivered to alveolar macrophages in the lungs of rodents. Guinea pig macrophages harvested by bronchoalveolar lavage were examined by differential interference contrast microscopy for morphological changes indicative of activation. Bronchoalveolar lavage fluid was analyzed for the presence of alkaline phosphatase, lactate dehydrogenase, N-acetyl-glucosaminidase (NAG), and total protein content. Rat alveolar macrophages were studied for the production of nitric oxide, by induction of nitric oxide synthase. Twenty-four hours following exposure to an aerosol of MDP, alveolar macrophages exhibited morphological characteristics (spreading and pseudopodia), enzyme activity (NAG 50% above control), and production of the reactive intermediate nitric oxide. Rat macrophages subjected to aerosol exposure to MDP when challenged with a second dose of MDP or lipopolysaccharide exhibited a linear dose response as measured by nitric oxide production. These studies indicate that the topical delivery of an MTB bacterial cell wall component, muramyl dipeptide, results in activation of alveolar macrophages. This approach may be useful in elucidating elements of the immune response to MTB.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1976

Effect of luteinizing hormone on the pattern of steroid production by preovulatory follicles of pregnant mare's serum gonadotropin-injected immature rats.

Preovulatory follicles were explanted on the day before ovulation from immature rats given a single injection of Pregnant Mare's Serum gonadotropin (PMS) 2 days earlier. The follicles were incubated for 4 h in modified Krebs bicarbonate buffer containing glucose and albumin in absence or presence of ovine luteinizing hormone (NIH-LH-S18; 0.1-10 mug/ml). The accumulation of progresterone, androstenedione and 17beta-estradiol in the medium was determined by radioimmunoassay. As in indicator of LH exposure the meiotic stage of the follicle-enclosed oocyte was determined at recovery by interference contrast microscopy. The first group of follicles were explanted in the morning, before the endogenous gonadotrophin surge. In hormone-free medium the oocytes remained in the dictyate stage, whereas addition of LH induced oocyte maturation. These follicles, when incubated in hormone-free medium, secreted predominantly androstenedione and estradiol and only low amounts of progesterone. In the presence of LH the secretion of all steroids was enhanced. The second group of follicles were explanted in the evening, 2-4 h after the endogenous gonadotrophin surge. After incubation in hormone-free medium the follicle-enclosed oocytes had matured. The steroid secretion by the follicles was different from that of the first group. In hormone-free medium they secreted predominantly progesterone and low amounts of androstenedione and estradiol. Addition of LH to the medium caused further enhancement of progesterone secretion, but had no effect on androstenedione and estradiol secretion. The third group of follicles were explanted in the evening from rats in which the preovulatory gonadotrophin surge had been prevented by Nembutal treatment. Oocyte maturation and steroid secretion did not differ from that found for the first group of follicles explanted in the morning. The results are compatible with the hypothesis that LH, after a transitory stimulation, inhibits androgen and estrogen secretion and stimulates progesterone secretion by the preovulatory ovarian follicle.

Androstenedione↗
PubMed2004

Gonadotropin-releasing hormone (GnRH) receptor expression and membrane signaling in early embryonic GnRH neurons: role in pulsatile neurosecretion.

The characteristic pulsatile secretion of GnRH from hypothalamic neurons is dependent on an autocrine interaction between GnRH and its receptors expressed in GnRH-producing neurons. The ontogeny and function of this autoregulatory process were investigated in studies on the properties of GnRH neurons derived from the olfactory placode of the fetal rat. An analysis of immunocytochemically identified, laser-captured fetal rat hypothalamic GnRH neurons, and olfactory placode-derived GnRH neurons identified by differential interference contrast microscopy, demonstrated coexpression of mRNAs encoding GnRH and its type I receptor. Both placode-derived and immortalized GnRH neurons (GT1-7 cells) exhibited spontaneous electrical activity that was stimulated by GnRH agonist treatment. This evoked response, as well as basal neuronal firing, was abolished by treatment with a GnRH antagonist. GnRH stimulation elicited biphasic intracellular calcium ([Ca2+]i) responses, and both basal and GnRH-stimulated [Ca2+]i levels were reduced by antagonist treatment. Perifused cultures released GnRH in a pulsatile manner that was highly dependent on extracellular Ca2+. The amplitude of GnRH pulses was increased by GnRH agonist stimulation and was diminished during GnRH antagonist treatment. These findings demonstrate that expression of GnRH receptor, GnRH-dependent activation of Ca2+ signaling, and autocrine regulation of GnRH release are characteristics of early fetal GnRH neurons and could provide a mechanism for gene expression and regulated GnRH secretion during embryonic migration.

Action Potentials↗
PubMed2005

Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.

Epithelial cells are equipped with junctional complexes that are involved in maintaining tissue architecture, providing mechanical integrity and suppressing tumour formation as well as invasiveness. A strict spatial segregation of these junctional complexes leads to the polarisation of epithelial cells. In vertebrate epithelia, basally localised hemidesmosomes mediate stable adhesion between epithelial cells and the underlying basement membrane. Although components of hemidesmosomes are relatively well known, the molecular machinery involved in governing the formation of these robust junctions, remains elusive. Here, we have identified the first component of this machinery using a forward genetic approach in zebrafish as we show that the function of penner (pen)/lethal giant larvae 2 (lgl2) is necessary for hemidesmosome formation and maintenance of the tissue integrity in the developing basal epidermis. Moreover, in pen/lgl2 mutant, basal epidermal cells hyper-proliferate and migrate to ectopic positions. Of the two vertebrate orthologues of the Drosophila tumour suppressor gene lethal giant larvae, the function of lgl2 in vertebrate development and organogenesis remained unclear so far. Here, we have unravelled an essential function of lgl2 during development of the epidermis in vertebrates.

Amino Acid Sequence↗
PubMed1988

Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.

In a search for molecular markers of male tail morphogenesis in C. elegans, we have detected two surface markers that are specifically observed in the copulatory bursa of adult males and the vulva of adult hermaphrodites. These markers are defined by binding of a monoclonal antibody (Ab117) and the lectin wheat germ agglutinin (WGA) to live intact animals. Expression of these markers is dependent on sex, stage and anterior-posterior position in the animal. Four of ten mutants with specific defects in bursal development show altered expression of one or both markers. Because the WGA marker can be expressed in intersexual animals with very little bursal development, posterior surface expression of this marker can serve as an indication of subtle masculinization of hermaphrodites. The timing of expression of these markers is not affected by heterochronic mutations that cause larval animals to express adult cuticles or adult animals to express larval cuticles, indicating that marker expression can be uncoupled from general cuticle development. Mutant lin-22 males, which have an anterior-to-posterior transformation of cell fates in the lateral hypodermis, ectopically express both markers in a manner consistent with a 'posteriorization' of positional information in these animals. These markers should be useful for the isolation and characterization of mutants defective in bursal and vulval development, sex determination and expression of anterior-posterior positional information.

Animals↗
PubMed1990

The micropyle: a sperm guidance system in teleost fertilization.

The micropylar region of the Rosy barb, Barbus conchonius, egg consists of 7-10 grooves and ridges, which drain directly into a funnel-shaped vestibule, the only point on the chorion through which sperm-egg contact is achieved during fertilization. Results of time-lapse video microscope study and computer-aided analysis of sperm motility pattern in the micropylar region showed that the fertilizing sperm, usually the first to enter the micropylar region, always travelled preferentially along the grooves into the micropylar pit. Subsequently, 86% of sperm arriving the micropylar region within 30 s travelled preferentially along the grooves into the immediate vicinity of the micropylar pit. The sperm guidance role of the micropylar region was calculated to enhance chances of egg penetration/fertilization by as much as 99.7% once sperm were within the micropylar region, possibly in response to some form of chemo-attractant(s) from the egg. Sperm agglutination post-fertilization was also found to occur preferentially along the grooves. Results of our in vitro fertilization experiments showed association between point of sperm entry and blastodisc formation: the blastodisc formed directly beneath the micropyle in all undisturbed eggs.

Animals↗
PubMed1991

Interactions of the Drosophila gap gene giant with maternal and zygotic pattern-forming genes.

The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development.

Animals↗
PubMed1999

Ontogeny and behaviour of early macrophages in the zebrafish embryo.

In the zebrafish embryo, the only known site of hemopoieisis is an intra-embryonic blood island at the junction between trunk and tail that gives rise to erythroid cells. Using video-enhanced differential interference contrast microscopy, as well as in-situ hybridization for the expression of two new hemopoietic marker genes, draculin and leucocyte-specific plastin, we show that macrophages appear in the embryo at least as early as erythroid cells, but originate from ventro-lateral mesoderm situated at the other end of the embryo, just anterior to the cardiac field. These macrophage precursors migrate to the yolksac, and differentiate. From the yolksac, many invade the mesenchyme of the head, while others join the blood circulation. Apart from phagocytosing apoptotic corpses, these macrophages were observed to engulf and destroy large amounts of bacteria injected intravenously; the macrophages also sensed the presence of bacteria injected into body cavities that are isolated from the blood, migrated into these cavities and eradicated the microorganisms. Moreover, we observed that although only a fraction of the macrophage population goes to the site of infection, the entire population acquires an activated behaviour, similar to that of activated macrophages in mammals. Our results support the notion that in vertebrate embryos, macrophages endowed with proliferative capacity arise early from the hemopoietic lineage through a non-classical, rapid differentiation pathway, which bypasses the monocytic series that is well-documented in adult hemopoietic organs.

Animals↗
PubMed2003

Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase.

Death-associated protein kinase (DAP kinase) is a proapoptotic, calcium/calmodulin-dependent serine/threonine kinase. Here, we report that DAP kinase phosphorylates the regulatory light chain of myosin II (MLC) both in vitro and in vivo, and that this phosphorylation occurs preferentially at residue Ser19. In quiescent fibroblasts, DAP kinase stabilizes stress fibers through phosphorylation of MLC, but it is dispensable for the formation of peripheral microfilament bundles. This cytoskeletal effect of DAP kinase occurs before the onset of apoptosis and does not require an intact death domain. In addition, DAP kinase is required for serum-induced stress-fiber formation, which is associated with the upregulation of its catalytic activity. Despite being both sufficient and necessary for the assembly or maintenance of stress fibers, DAP kinase is incapable of stimulating the formation of focal adhesions in quiescent cells. Moreover, it promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Together, our results identify a novel and unique function of DAP kinase in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity.

3T3 Cells↗
PubMed2004

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation.

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

3T3 Cells↗
PubMed2004

Dynamics of novel feet of Dictyostelium cells during migration.

We observed the dynamics of actin foci in live Dictyostelium cells expressing GFP-actin. Actin foci were dynamic structures, but they were fixed on the substratum during cell migration. Interference reflection microscopy revealed that the ventral cell membrane was closer to the substratum at sites of actin foci. Furthermore, some actin foci were incorporated into the retraction fibers, ripped off from the cells and eventually shed on the substratum after the cells moved away. The velocity of the cells was inversely proportional to the number of actin foci. Measurement of traction force using a silicone substratum demonstrated that the traction force was transmitted to the substratum through actin foci. Taken together, several lines of evidence strongly suggest that actin foci function as the active 'feet' of Dictyostelium cells. We also found evidence suggesting that changing step is regulated in a coordinated manner during cell migration. Possible mechanisms by which these cells migrate across substrata are discussed in this context.

Actins↗