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Expression of ras cellular oncogenes during the early postimplantation period in the rat was investigated using immunohistochemistry to p21ras. A broad spectrum polyclonal antibody recognizing N-, Ha- and Ki- forms of p21ras was used in an indirect avidin-biotin-peroxidase (ABC) technique. Positive staining indicating the presence of p21ras was found in embryos from 6.5 to 12 days embryonic age. In early egg cylinders (6.5 days), positive staining for p21ras was observed on the ectoplacental cone, primitive ectoderm and trophectoderm, while primitive endoderm and parietal endoderm appeared paler. In later egg cylinder stages (7.5 days), strong positive staining was observed in the primitive embryonic ectoderm and ectoplacental cone, but parietal and visceral endoderm still appeared to be devoid of positive staining. As development proceeded during primitive streak stages, the visceral and parietal endoderm became positively stained. By 10 days, all tissues appeared to be positive for p21ras, with strong staining appearing in the heart and neural elements. Therefore, p21ras does not appear to be ubiquitous in the rat conceptus prior to gastrulation, but shows differential distribution, appearing later in endodermal derivatives. Possibly p21ras is involved in determination of the ectodermal and endodermal lineages.
Anat Rec 1992 Nov
PMID:Distribution of p21ras in postimplantation rat embryos. 144 70

The presence of proteoglycans (PGs) was studied in compact lamellar rat and human bone at the electron microscopic level. With the cationic dye cuprolinic blue (CB1), PGs could be demonstrated in the mineralized bone matrix. The amounts of PGs appeared to be equal in the different lamellae and osteons. More CBl-positive material was found in the outermost lamella of the cortex, in the perilacunar matrix around the osteocyte lacunae, and around the canaliculi. Enzyme digestion with chondroitinase ABC demonstrated that the CBl-positive rods consisted of PGs. These observations amplify biochemical studies in which PGs have been isolated from the mineralized bone matrix. The presence of CBl-positive rods in the mineralized matrix suggest that PGs do not have to be removed completely to make the matrix calcifiable.
Anat Rec 1992 Jan
PMID:An electron microscopic study on the presence of proteoglycans in the mineralized matrix of rat and human compact lamellar bone. 153 63

We have studied the distribution of anionic sites in the basal lamina of developing human amniotic epithelium by using the cationic stain ruthenium red. Amnions at 7-12 weeks of gestation and at term contained ruthenium red-positive granules in a quasi-regular array on both the cellular and interstitial sides of the lamina densa. In order to characterize the anionic sites, small pieces of amnion were incubated in the presence or absence of either chondroitinase ABC, neuraminidase, Streptomyces hyaluronidase, or heparitinase in appropriate buffer systems. Incubation in the presence of heparitinase resulted in the complete disappearance of the basal lamina-associated granules, but other enzymes tested had no demonstrable effect on these granules. We conclude that the anionic sites associated with amnion basal lamina, and demonstrable with ruthenium red, consist of glycosaminoglycans rich in heparan sulfate, probably present as heparan sulfate proteoglycan. Because amniotic fluid has a low protein content and amniotic epithelium (at least at term) lacks tight junctions, we postulate that the heparan sulfate proteoglycan associated with the amnion basal lamina may have an important function as a permeability barrier to anionic macromolecules.
Anat Rec 1985 May
PMID:Distribution and characterization of anionic sites in the basal lamina of developing human amniotic epithelium. 241 50

Human NK activity is known to be associated with a population of large granular lymphocytes (LGL) exhibiting several immunophenotypic surface markers including Leu-11a (NKP-15), Leu-7 (HNK-1), Leu-3a (T4), and Leu-2a (T8). Based upon correlation with cytolytic activity, Leu-11a is now considered the most specific antigenic marker for human NK cells. Present investigation compared the ultrastructure of cells expressing Leu-11a, Leu-7, Leu-3a, and Leu-2a, both in human peripheral blood lymphocytes (PBL) and the purified LGL fraction. Subcellular cytochemical reactions were investigated in Leu-7+ or Leu-11a+ PBL or LGL and in cells conjugated with K562 targets (indicating NK cytolytic potential). The surface markers, localized with monoclonal antibodies, were detected by immunoelectron microscopy by using direct or indirect avidin-biotin-peroxidase (ABC) or colloidal gold methods. A peroxidase-colloidal gold double-labeling system was used to identify subsets of Leu-7+ or Leu-11a+ cells. Previously described ultrastructural features of LGL including a villous surface, reniform nuclei, low nuclear/cytoplasm ratios, and abundant cytoplasm with vesicles, vacuoles, electron-dense granules, parallel tubular arrays (PTA), or paracrystalline inclusions were associated with Leu-7+, Leu-11a+, Leu-7+/Leu-11a+, Leu-7+/Leu-11a-, and Leu-7-/Leu-11a+ PBL or LGL. Results showed that the Leu-7+/Leu-11a+ cells were the most abundant NK cells in PBL. Lymphocyte subsets with Leu-3a or Leu-2a surface marker showed some ultrastructural features including PTA similar to Leu-7+ cells and Leu-11a+ cells, and their subsets. These T-cells appeared ultrastructurally more similar to the Leu-7+/Leu-11a- subset. Cytochemical studies showed that electron-dense cytoplasmic granules and PTA typical of the Leu-11a+ cells and Leu-7+ cells contained glycoprotein, acid phosphatase, and arylsulfatase. Large cytoplasmic vacuoles were heterogeneous and typically contained electron-dense material with DAB reactivity, membranous material, PTA, and/or paracrystalline inclusions. Glycoprotein, acid phosphatase, and arylsulfatase, and peroxidase reactive material were also found in these vacuoles. These features suggested that the vacuoles could be secondary lysosomes. The coexistence of intact PTA or degenerating PTA in the same vacuoles with paracrystalline inclusions suggested that the latter are possibly derived from PTA.
Anat Rec 1987 Mar
PMID:Immunoultrastructural studies of human NK cells: I. Ultracytochemistry and comparison with T cell subsets. 355 61

The distribution of anionic sites was studied in the trophoblastic and fetal capillary basal laminas of developing human placental villi with the cationic stain ruthenium red. At 7-12 weeks of gestation the trophoblastic basal lamina (TBL) contained ruthenium red-positive granules in a quasi-regular array throughout the lamina densa or sometimes concentrated at the interstitial surface of the lamina densa. The capillary basal lamina (CBL) (and anionic sites) were not present at this age. Anionic sites were also associated with collagen or reticular fibrils. At term, the TBL was largely devoid of anionic sites except for some distributed along its interstitial surface. The CBL was present in later gestation and sometimes had arrays of anionic sites. In order to characterize the anionic sites, minced pieces of villi were incubated in the presence or absence of either chondroitinase ABC, heparitinase, neuraminidase, or Streptomyces hyaluronidase in appropriate buffer systems. Incubation of early villi with heparitinase resulted in the disappearance of the TBL-associated sites. Chondroitinase ABC appeared to reduce staining of collagen-associated sites. In term villi, heparitinase removed those few sites still associated with the TBL but did not affect sites associated with the CBL or collagen. Chondroitinase ABC resulted in the disappearance of all anionic sites. In later gestation, a number of developmentally important macromolecules are transported across the trophoblast and enter the fetal capillaries. We conclude that the absence of an array of polyanionic sites from the term placenta TBL and the reduction in the amount of extracellular matrix intervening between the trophoblast and capillaries are adaptations to enhance the exchange of macromolecules across the placenta.
Anat Rec 1985 May
PMID:Distribution and characterization of anionic sites in trophoblast and capillary basal laminas of human placental villi. 407 43

Sulfated glycoconjugates were stained in normal human term placentas using Spicer's high-iron diamine (HID) method with thiocarbohydrazide and silver proteinate (TCH-SP) enhancement. Specific identification of glycosaminoglycans (GAG) was accomplished by digestion of the stained material with chondroitinase ABC or AC for removal of chondroitin sulfates and nitrous acid for removal of N-sulfated GAGs. The syncytiotrophoblast apical surface demonstrated moderate to intense staining with HID-TCH-SP, which was removed by prior digestion with the chondroitinases, but not by nitrous acid. The syncytiotrophoblast basal surface and endothelial cell surfaces lacked sulfate staining. A few cytoplasmic granules in syncytiotrophoblast cells demonstrated staining similar to the apical surface. Three layers of the basal lamina were identified in these preparations. The lamina lucida immediately beneath the syncytiotrophoblast and the majority of the lamina densa stained weakly or not at all, whereas the underlying lamina diffusa and stroma demonstrated moderate to intense staining. The majority of lamina diffusa staining was removed by chondroitinase ABC or AC; the remaining material was removed by nitrous acid digestion. Thus the syncytiotrophoblast surface contains a chondroitin sulfate and the basal lamina contains a mixture of intensely stained chondroitin sulfate and a weakly stained N-sulfated GAG.
Anat Rec 1984 Nov
PMID:Ultrastructural localization of glycosaminoglycans in human term placenta. 608 29

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-Mr rec-TM), 0.11 nM thrombin inactivated 50% of 4.4 nM scu-PA in 45 min at 37 degrees C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-Mr rec-TM), 1.9 nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7 nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4 nM for high-Mr rec-TM and 14 nM for low-Mr rec-TM. Treatment of high-Mr rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-Mr rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-Mr rec-TM. The free glycosaminoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex.
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PMID:Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin. 838 42

Laboratory investigations into equivalence class formation suggest how animals in social and communicative contexts learn to place dissimilar individuals, signals, responses and social reinforcers into the same functional class. Kastak & Schusterman (1994, Anim. Learn. Behav., 22, 427-435) demonstrated that a California sea lion performed generalized identity matching-to-sample; that is, it chose visual stimulus A conditionally upon an identical sample A (AA matching), chose stimulus B conditionally upon sample B (BB matching) and chose stimulus C conditionally upon sample C (CC matching). The sea lion was later trained on 30 problems with similar stimuli to select comparison B conditionally upon sample A (AB matching), and trained on another 30 problems to select comparison C conditionally upon sample B (BC matching). Subsequently, the sea lion demonstrated trial-1 BA and CB matching and trial-1 AC and CA matching (Schusterman & Kastak 1993, Psychol. Rec., 43, 823-839). Matching of these derived relations defines the phenomenon of stimulus equivalence: when one member (A) of an equivalence class (ABC) becomes discriminative for a given behaviour, then B and C should become discriminative for the same behaviour. In the current study, we tested whether the sea lion could transfer the relations it had acquired between equivalence class members from a matching-to-sample paradigm to a simple discrimination paradigm. In 28 of 30 tests, the sea lion immediately transferred the discriminative function acquired by one member of an equivalence class to the remaining members of that class. Substitutability among members of an equivalence class is relevant to an analysis of referential communication, for example, the representational function of alarm calls. Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.
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PMID:Functional equivalence in a California sea lion: relevance to animal social and communicative interactions. 963 96

The extensor tendons of the fingers and toes form part of the capsule of the interphalangeal joint and press against the proximal phalanx during flexion. Previous work on the fingers has shown that there is a "sesamoid" fibrocartilage on the deep surface of each tendon that labels immunohistochemically for a variety of glycosaminoglycans and collagens. However, we know little about the molecular composition of the tendon in the toes. This question is of special interest, because the mechanics of the interphalangeal joints differ in the upper and lower limbs-the toes balance the forefoot, distribute load during the gait cycle, and transmit the pull of larger muscles. This means that their extensor tendons are more often under higher tension than those in the fingers. Here, we report the presence of an equivalent fibrocartilage and compare its immunolabelling characteristics in all the toes. Six forefeet were removed from elderly cadavers, and the interphalangeal (IP) joints were fixed in 90% methanol. The extensor tendon and its enthesis were dissected out from the IP joint of the big toe and from the proximal interphalangeal (PIP) joint of all lesser toes, decalcified, cryosectioned, and immunolabelled with a panel of monoclonal and polyclonal antibodies for type I, II, III, and VI collagens; chondroitin 4 and 6 sulphates; and dermatan and keratan sulphate. Antibody binding was detected with the Vectastain ABC Elite avidin-biotin-peroxidase kit (Vector Laboratories, Burlingame, CA). The extensor tendon in all the toes had a metachromatic, sesamoid fibrocartilage on its deep surface that immunolabelled for all glycosaminoglycans and for type I, III, and VI collagens. Labelling for type II collagen was seen in the sesamoid fibrocartilage of all toes but was particularly characteristic of the 2nd through 5th toes. The immunolabelling patterns of the enthesis fibrocartilage were similar in all toes and to results reported previously for fingers. The normal occurrence of type II collagen in the sesamoid fibrocartilage of the 2nd through 5th toes is in contrast to our published data on the fingers. The finding can be related to the more constant loading of the tendon in the toes. The greater prominence of type II collagen in the sesamoid fibrocartilage of the 2nd through 5th toes could be related to a difference in joint position during walking between the 1st toe and the 2nd through 5th toes--the PIP joints of the latter are usually more flexed than the IP joint of the former.
Anat Rec 1998 10
PMID:Fibrocartilages in the extensor tendons of the interphalangeal joints of human toes. 977 80

Galanin is a brain-gut peptide that is present in the central and peripheral nervous systems. In the gut, it is contained exclusively in intrinsic and extrinsic nerve supplies, and it is involved overall in the regulation of gut motility. To obtain information about the ontogeny of galanin, we undertook an immunohistochemical study of chicken embryos. The time of first appearance and the distribution patterns of galanin were investigated with fluorescence and streptavidin-biotin-peroxidase (ABC) immunohistochemical protocols by using a galanin polyclonal antiserum. The various regions of the gut and the pancreas were obtained from chicken embryos aged from 3 days of incubation to hatching. All specimens were fixed in buffered picric acid-paraformaldehyde, frozen, and cut with a cryostat. Galanin-immunoreactive neuroblasts were first detected at 4 days in the mesenchyme of the proventriculus/gizzard primordium and within the Remak ganglion. They then extended cranially and caudally, reaching all of the other gut regions at 6.5 days. Galanin-immunoreactive nerve elements mainly occupied the sites of myenteric and submucous plexuses. From day 15, galanin-immunoreactive nerve fibers tended to invade the circular muscular layer and part of the lamina propria of the mucosa. In the pancreas, weak galanin-immunoreactive nerve elements were detected at 5.5 days. They tended to be distributed among the glandular lobules according to the organ differentiation. The widespread distribution during the earlier embryonic stages represents evidence indicating that the neuropeptide galanin may have a role as a differentiating or growth factor. From late embryonic life, its predominant presence in sympathetic nerves and in muscular layers fits with the functions demonstrated previously in adults of other vertebrates for galanin as a modulator of intestinal motility.
Anat Rec 1999 01
PMID:Ontogeny of galanin-immunoreactive elements in the intrinsic nervous system of the chicken gut. 989 15


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