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  • Oligodendrocyte dysfunction after induction of experimental anterior optic nerve ischemia. 16043843

    The early response and survival of oligodendrocytes after axonal stroke and their potential contribution to neuronal survival in vivo have not been adequately addressed. The purpose of this study was to investigate the changes occurring in the retina and optic nerve (ON) in anterior ischemic optic neuropathy (AION), using a c-fos transgenic mouse model.A new mouse model of AION (rodent AION) was developed to evaluate the in vivo stress response of oligodendrocytes and retinal ganglion cells (RGCs) in a transgenic mouse strain, using the immediate early stress-response gene c-fos, RT-QPCR technology, immunohistochemistry, and electron microscopy. Confocal microscopy was used with cell-specific antibodies to characterize the timing of cells responding to rAION. The TUNEL assay detected cells undergoing apoptosis. Ultrastructural changes were analyzed by electron microscopy.In rAION, oligodendrocytes rapidly respond in vivo to ischemic ON damage, with c-fos activation as an early detectable event. Early evidence of progressive oligodendrocyte stress, is followed by demyelination, wallerian degeneration of the ON, and oligodendrocyte and RGC death far from the primary lesion.After rAION induction oligodendrocytes, as well as RGCs, undergo progressive stress, with dysfunction and apoptosis. The findings lead to a proposal that progressive retrograde oligodendrocyte stress, away from the primary lesion, is an important factor after ischemic optic neuropathy. Postinduction demyelination must be addressed for effective neuroprotection of ischemic and hypoxic white matter.
    Document Type:
    Reference
    Product Catalog Number:
    MAB342
    Product Catalog Name:
    Anti-Galactocerebroside Antibody, clone mGalC

  • Human placenta type V collagens. Evidence for the existence of an alpha 1(V) alpha 2(V) alpha 3(V) collagen molecule. 6501291

    Human type V collagen was purified from placenta and found to contain alpha 1(V), alpha 2(V), and alpha 3(V) chains in varying ratios. Using any of three independent nondenaturing methods (phosphocellulose chromatography, high-performance ion-exchange chromatography on IEX-540 DEAE, and ammonium sulfate precipitation), this preparation could be resolved into two fractions. Analysis of the two fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that one fraction contained alpha 1(V) and alpha 2(V) in a 2:1 ratio and the other contained alpha 1(V), alpha 2(V), and alpha 3(V) in a 1:1:1 ratio. When the crude placental type V collagen was electrophoresed under nondenaturing conditions, two bands were observed, one co-migrating with purified (alpha 1(V]2 alpha 2(V) and the other co-migrating with the fractions containing alpha 1(V), alpha 2(V), and alpha 3(V) chains in a 1:1:1 ratio. Electrophoresis in a second dimension under denaturing conditions confirmed that the fast-migrating band contained (alpha 1(V]2 alpha 2(V) and that the slow-migrating band contained the three chains in equimolar ratio. CD spectra of the two fractions and resistance to trypsin-chymotrypsin digestion confirmed that the two fractions contain triple helical collagen. Thermal denaturations were monitored by the changes in CD signal at 221 nm. The two fractions purified by ammonium sulfate precipitation melted at 39.1 and 36.4 degrees C for the (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) fractions, respectively. Trypsin cleavage of these two native fractions at temperatures near melting produced completely different fragmentation patterns, indicating different partial unwinding sites of the alpha 1(V) and alpha 2(V) chains in the two preparations and thus different molecular assemblies. Our data demonstrate the existence of two different molecular assemblies of type V collagen in human placenta consisting of (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) heterotrimers.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3302

  • PREPARATION OF A CAPSAICIN-LOADED NANOEMULSION FOR IMPROVING SKIN PENETRATION 24417234

    Capsaicin o/w nanoemulsions with enhanced skin permeation were successfully prepared by controlling the ratios of the surfactant mixtures, oleoresin capsicum as the oil phase, and aqueous phase. Oleoresin capsicum contains 22.67 mg/g of capsaicin, which is an active and oil-soluble ingredient. Nonionic surfactants, Tween 80 and Span 80, were used to optimize the weight ratio of surfactant mixtures (85.98:14.02) by calculating the hydrophile-lipophile balance (HLB) value. The optimal processing conditions for stable nanoemulsions were investigated by using a ternary phase diagram. The mean droplet size of nanoemulsions ranged from 20 to 62 nm. Skin permeation studies were performed using a Franz diffusion cell. The permeation profiles and confocal laser scanning microscopy (CLSM) images supported that capsaicin nanoemulsion could well permeate all skin layers from the stratum corneum to the dermis. The selected nanoemulsions showed great potential as transdermal delivery carriers for enhancing the permeation of core materials.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear re ... 1704480

    We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.
    Document Type:
    Reference
    Product Catalog Number:
    ABE1424
    Product Catalog Name:
    Anti-Glucocorticoid Receptor Antibody

  • Postprandial response of plasma insulin, amylin and acylated ghrelin to various test meals in lean and obese cats. 20100379

    The propensity of diets of different composition to promote obesity is a current topic in feline medicine. The effects of three meals with different protein:fat ratios on hormones (insulin, acylated ghrelin and amylin) involved in the control of food intake and glucose metabolism were compared. Five lean (two females and three males, 28.6 (sd 3.4) % body fat mass (BFM), mean body weight (BW) 4590 g) and five obese (two females and three males, 37.1 (sd 4.1) % BFM, mean BW 4670 g) adult cats were studied. Only BFM differed significantly between obese and lean cats. The cats were fed a high-protein (HP), a high-fat and a high-carbohydrate diet in a randomised cross-over design. Food intake did not differ between cats fed on the different diets, but obese cats consumed significantly more energy, expressed as per kg fat-free mass, than lean cats. After a 6-week adaptation period, a test meal was given and blood samples were collected before and 0, 30, 60 and 100 min after the meal. Baseline concentrations of glucose, amylin and acylated ghrelin were higher in obese cats than in lean cats, and obese cats showed the highest postprandial responses of glucose and amylin. The HP diet led to higher postprandial amylin concentrations than the other diets, indicating a possible effect of amino acids on beta-cell secretion. Postprandial ghrelin concentrations were unaffected by diet composition. The relationship between insulin, amylin and ghrelin secretion and their relevant roles in food intake and glucose metabolism in cats require further study.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Inducible coexpression of connexin37 or connexin40 with connexin43 selectively affects intercellular molecular transfer. 22729648

    Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 μM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3068

  • Zebrafish mnx1 controls cell fate choice in the developing endocrine pancreas. 21989909

    The vertebrate endocrine pancreas has the crucial function of maintaining blood sugar homeostasis. This role is dependent upon the development and maintenance of pancreatic islets comprising appropriate ratios of hormone-producing cells. In all vertebrate models studied, an initial precursor population of Pdx1-expressing endoderm cells gives rise to separate endocrine and exocrine cell lineages. Within the endocrine progenitor pool a variety of transcription factors influence cell fate decisions, such that hormone-producing differentiated cell types ultimately arise, including the insulin-producing beta cells and the antagonistically acting glucagon-producing alpha cells. In previous work, we established that the development of all pancreatic lineages requires retinoic acid (RA) signaling. We have used the zebrafish to uncover genes that function downstream of RA signaling, and here we identify mnx1 (hb9) as an RA-regulated endoderm transcription factor-encoding gene. By combining manipulation of gene function, cell transplantation approaches and transgenic reporter analysis we establish that Mnx1 functions downstream of RA within the endoderm to control cell fate decisions in the endocrine pancreas progenitor lineage. We confirm that Mnx1-deficient zebrafish lack beta cells, and, importantly, we make the novel observation that they concomitantly gain alpha cells. In Mnx1-deficient embryos, precursor cells that are normally destined to differentiate as beta cells instead take on an alpha cell fate. Our findings suggest that Mnx1 functions to promote beta and suppress alpha cell fates.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Role of the programmed Death-1 pathway in the suppressive activity of alternatively activated macrophages in experimental cysticercosis. 16126211

    We characterised a population of macrophages potentially involved in the immunoregulation induced by experimental cysticercosis. Following Taenia crassiceps infection, macrophages recruited in the peritoneal cavity were isolated and co-cultured at different ratios with T cells from naïve mice previously stimulated with anti-CD3/CD28 antibodies; these macrophages inhibited naïve T cell proliferation. This suppressive effect was Interleukin (IL)-10, Interferon-gamma (IFN-gamma), and nitric oxide (NO) independent. In contrast, macrophage-T cell contact was necessary to maintain anergy of T cells. Reverse transcriptase-PCR analysis of these macrophages showed higher transcripts of IL-10, chitinases Fizz1 and Ym1, and arginase-1 compared with naïve macrophages; by contrast, IL-12p40, and inducible nitric oxide synthase (iNOS) transcripts were undetected, whereas C-C chemokine ligand 5 (CCL5) was unchanged. Analysis of the membrane molecules expressed on Taenia-induced macrophages showed an up-regulation of several markers, mainly programmed death ligand 1 (PD-L1) and PD-L2. Blockade of PD-L1, PD-L2 or their receptor PD-1, but not of another marker, eliminated their ability to inhibit T-cell proliferation. Parallel experiments using ovalbumin (OVA)-peptide as a model antigen displayed similar results. Additionally, the same mechanism appears to be functional in splenocytes of T. crassiceps-infected mice given that blockade of PD-1, PD-L1 or PD-L2 re-established their ability to proliferate in response to parasite antigens. Moreover, Taenia-induced macrophages were able to suppress a mixed lymphocyte reaction in a PD-1-dependent manner. Thus, cestode infections induce macrophages alternatively activated with strong suppressive activity involving the PD-1/PD-L's pathway.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Bone marrow-derived mesenchymal stem cells modulate BV2 microglia responses to lipopolysaccharide. 20850581

    The immunoregulatory properties of mesenchymal stem cells (MSC) have been demonstrated on a wide range of cells. Here, we describe the modulatory effects of mouse bone marrow-derived MSC on BV2 microglia proliferation rate, nitric oxide (NO) production and CD40 expression. Mouse bone marrow MSC were co-cultured with BV2 cells at various seeding density ratios and activated with lipopolysaccharide (LPS). We show that MSC exert an anti-proliferative effect on microglia and are potent producers of NO when stimulated by soluble factors released by LPS-activated BV2. MSC suppressed proliferation of both untreated and LPS-treated microglia in a dose-dependent manner, significantly reducing BV2 proliferation at seeding density ratios of 1:0.2 and 1:0.1 (p<.05). Co-culturing MSC with BV2 cells at different ratios revealed interesting dynamics in NO production. A high number of MSC significantly increases NO in co-cultures whilst a lower number reduces NO. The increased NO levels in co-cultures may be MSC-derived, as we also show that activated BV2 cells stimulate MSC to produce NO. Cell-cell interaction is not a requirement for this effect as soluble factors released by activated BV2 cells alone do stimulate MSC to produce high levels of NO. Although NO is implicated as a mediator for T cell proliferation, it does not appear to play a major role in the suppression of microglia proliferation. Additionally, MSC reduced the expression of the microglial co-stimulator molecule, CD40. Collectively, these regulatory effects of MSC on microglia offer insight into the potential moderating properties of MSC on inflammatory responses within the CNS.
    Document Type:
    Reference
    Product Catalog Number:
    SCR020
    Product Catalog Name:
    Mesenchymal Adipogenesis Kit

  • Effects of glucose-to-fructose ratios in solutions on subjective satiety, food intake, and satiety hormones in young men. 17991646

    BACKGROUND: The greater prevalence of obesity and the metabolic syndrome in the past 35 y has been attributed to the replacement of sucrose in the food supply with high-fructose corn syrup (HFCS). OBJECTIVE: Two experiments were conducted to determine the effect of solutions containing sucrose, HFCS, or various ratios of glucose to fructose (G:F) on food intake (FI), average appetite (AA), blood glucose (BG), plasma insulin, ghrelin, and uric acid (UA) in men. DESIGN: Sugar solutions (300 kcal/300 mL) were (in %) G20:F80, HFCS 55 (G45:F55), sucrose, and G80:F20 (experiment 1, n = 12) and G20:F80, G35:F65, G50:F50, sucrose, and G80:F20 (experiment 2, n = 19). The controls were a sweet energy-free control (experiment 1) and water (both experiments). Solutions were provided in a repeated-measures design. AA, BG, and FI were measured in all subjects. Hormonal responses and UA were measured in 7 subjects in experiment 2. Measurements were taken from baseline to 75 min. FI was measured at 80 min. RESULTS: Sucrose and HFCS (experiment 1) and sucrose and G50:F50 (experiment 2) had similar effects on all dependent measures. All sugar solutions similarly reduced the AA area under the curve (AUC). FI and plasma UA concentrations were significantly (P 0.05) lower after high-glucose solutions than after low-glucose solutions. The lower FI was associated with a greater BG AUC (P 0.05) and smaller AA and ghrelin AUCs (P 0.01). Insulin and BG AUCs were positively associated (P 0.001). CONCLUSION: Sucrose, HFCS, and G50:F50 solutions do not differ significantly in their short-term effects on subjective and physiologic measures of satiety, UA, and FI at a subsequent meal.
    Document Type:
    Reference
    Product Catalog Number:
    GHRT-89HK
    Product Catalog Name:
    Human Ghrelin (TOTAL) RIA