Structure and function of the selectin ligand PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric mucin-like 120-kDa glycoprotein on leukocyte surfaces that binds to P- and L-selectin and promotes cell adhesion in the inflammatory response. The extreme amino terminal extracellular domain of PSGL-1 is critical for these interactions, based on site-directed mutagenesis, blocking monoclonal antibodies, and biochemical analyses. The current hypothesis is that for high affinity interactions with P-selectin, PSGL-1 must contain O-glycans with a core-2 branched motif containing the sialyl Lewis x antigen (NeuAca2®3Galß1®4[Fuca1®3]GlcNAcß1®R). In addition, high affinity interactions require the co-expression of tyrosine sulfate on tyrosine residues near the critical O-glycan structure. This review addresses the biochemical evidence for this hypothesis and the evidence that PSGL-1 is an important in vivo ligand for cell adhesion.

Effect of SX-3228, a selective ligand for the BZ1 receptor, on sleep and waking during the light-dark cycle in the rat

The effects of the benzodiazepine1 (BZ1) receptor agonist SX-3228 were studied in rats (N = 12) implanted for chronic sleep procedures. Administration of 0.5, 1.0 and 2.5 mg/kg SX-3228, sc, to rats 1 h after the beginning of the light phase of the light-dark cycle induced a significant reduction of rapid-eye-movement sleep (REMS) during the third recording hour. Moreover, slow wave sleep (SWS) was increased during the fourth recording hour after the two largest doses of the compound. Administration of 0.5, 1.0 and 2.5 mg/kg SX-3228 one hour after the beginning of the dark period of the light-dark cycle caused a significant and maintained (6-h recording period) reduction of waking (W), whereas SWS and light sleep (LS) were increased. REMS values tended to increase during the entire recording period; however, the increase was statistically significant only for the 1.0 mg/kg dose during the first recording hour. In addition, a significant and dose-related increase of power density in the delta and the theta regions was found during nonREM sleep (LS and SWS) in the dark period. Our results indicate that SX-3228 is a potent hypnotic when given to the rat during the dark period of the light-dark cycle. Moreover, the sleep induced by SX-3228 during the dark phase closely resembles the physiological sleep of the rat.

Expression of CD40 ligand, interferon-gamma and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection

The purpose of the present study was to investigate the expression (mRNA) of CD40 ligand (CD40L), interferon-gamma (IFN-gamma) and Fas ligand (FasL) genes in human cardiac allografts in relation to the occurrence of acute cardiac allograft rejection as well as its possible value in predicting acute rejection. The mRNA levels were determined by a semiquantitative reverse transcriptase-polymerase chain reaction method in 39 samples of endomyocardial biopsies obtained from 10 adult cardiac transplant recipients within the first six months after transplantation. Biopsies with ongoing acute rejection showed significantly higher CD40L, IFN-gamma and FasL mRNA expression than biopsies without rejection. The median values of mRNA expression in biopsies with and without rejection were 0.116 and zero for CD40L (P<0.003), 0.080 and zero for IFN-gamma (P<0.0009), and 0.156 and zero for FasL (P<0.002), respectively. In addition, the levels of IFN-gamma mRNA were significantly increased 7 to 15 days before the appearance of histological evidence of rejection (median of 0.086 in pre-rejection biopsies), i.e., they presented a predictive value. This study provides further evidence of heightened expression of immune activation genes during rejection and shows that some of these markers may present predictive value for the occurrence of acute rejection.

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

Effect of iodide on Fas, Fas-ligand and Bcl-w mRNA expression in thyroid of NOD mice pretreated with methimazole

Nonobese diabetic (NOD) mice and a derived strain, NOD.H.2h4, have been used as a model for experimental spontaneous thyroiditis and thyroiditis induced by iodide excess after a goiter-inducing period. Some authors have proposed that iodide, given after methimazole or propylthiouracil, is capable of inducing apoptosis in thyroid cells and that anti-thyroid drugs can modulate the expression of apoptosis components such as Fas and its ligand (Fas-L). Here we evaluated the effect of potassium iodide (20 µg/animal for 4 days, ip) given to NOD mice at the 10th week of life after exposure to methimazole (1 mg/ml) in drinking water from the 4th to the 10th week of life. Fas, Fas-L and Bcl-w expression were analyzed semiquantitatively by RT-PCR immediately after potassium iodide administration (group MI44D) or at week 32 (MI32S). Control groups were added at 10 (C10) and 32 weeks (C32), as well as a group that received only methimazole (CM10). An increase in the expression of Fas-L and Bcl-w (P<0.01, ANOVA) was observed in animals of group MI44D, while Fas was expressed at higher levels (P = 0.02) in group C32 (72.89 ± 47.09 arbitrary units) when compared to group C10 (10.8 ± 8.55 arbitrary units). Thus, the analysis of Fas-L and Bcl-w expression in the MI44D group and Fas in group C32 allowed us to detect two different patterns of expression of these apoptosis components in thyroid tissue of NOD mice.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

Notch ligand Delta-like 1 promotes the metastasis of melanoma by enhancing tumor adhesion

Notch signaling plays a vital role in tumorigenicity and tumor progression by regulating proliferation, invasion, and the tumor microenvironment. Previous research by our group indicated that Notch ligand Delta-like 1 (Dll1) is involved in angiogenesis in melanoma, and we noticed that it took a longer time to trypsinize Dll1-expressing B16 melanoma cells than the control cells. In this article, we extended our study to investigate the effects of Dll1 on tumor cell adhesion and metastasis. Dll1 overexpression activated Notch signaling in B16 tumor cells and significantly enhanced the adhering capacity of B16 tumor cells both in vitro and in vivo. B16-Dll1 cells also had a higher metastatic potential than their counterpart in the mouse model of lung metastasis. Along with increased Dll1 expression, N-cadherin, but not E-cadherin, was upregulated in B16-Dll1 cells. These data suggested that Notch ligand Dll1 may enhance the adhesion and metastasis of melanoma cells by upregulation of N-cadherin.

Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.

Eficiência de um kit de ELISA na detecção e quantificação de aflatoxina M1 em leite e investigação da ocorrência no estado de Minas Gerais

Procedimentos para validação intralaboratorial de kits de ELISA foram adotados na verificação da eficiência de um kit para detecção e quantificação de aflatoxina M1 em leite. Foram realizados ensaios com soluções padrões fornecidas pelo kit, amostras artificialmente contaminadas e amostras naturalmente contaminadas. Os valores médios de recuperação obtidos para os padrões do kit apresentaram-se entre 85,6 e 114,8%, com valores de coeficiente de variação entre 11,6 e 23,1%. Nos ensaios com amostras artificialmente contaminadas, foi definida uma faixa ótima de trabalho para análises quantitativas entre 0,019 e 0,090µg/L. A presença de aflatoxina M1 em 110 amostras de leite do estado de Minas Gerais foi investigada. Cinco das 27 amostras positivas na triagem por ELISA foram confirmadas por cromatografia em camada delgada.