High total cholesterol and triglycerides levels increase arginases metabolism, impairing nitric oxide signaling and worsening fetoplacental endothelial dysfunction in gestational diabetes mellitus pregnancies.

Maternal physiological dyslipidemia (MPD) supports fetal development in human pregnancy. However, some women develop maternal supraphysiological dyslipidemia (MSPD: increased total cholesterol (TC) and triglycerides (TG) levels). MSPH is present in normal and also in gestational diabetes mellitus (GDM) pregnancies. MSPD and GDM associate with fetoplacental endothelial dysfunction, producing alterations in nitric oxide (NO)-L-arginine/arginase metabolism. Nevertheless, the effect of MSPD on GDM, and how this synergy alters fetoplacental endothelial function is unknown. Therefore, the aim of this study was to evaluate in human umbilical vein endothelial cells, the effects of MSPD in GDM and how these pathologies together affect the fetoplacental endothelial function. 123 women at term of pregnancy were classified as MPD (n = 40), MSPD (n = 35), GDM with normal lipids (GDM-MPD, n = 23) and with increased lipids (GDM-MSPD, n = 25). TC ≥291 mg/dL and TG ≥275 mg/dL were considered as MSPD. Endothelial NO synthase (eNOS), human cationic amino acid transporter 1 (hCat1), and arginase II protein abundance and activity, were assayed in umbilical vein endothelial cells. In MSPD and GDM-MSPD, TC and TG increased respect to MPD and GDM-MPD. eNOS activity was reduced in MSPD and GDM-MSPD, but increased in GDM-MPD compared with MPD. However, decreased tetrahydrobiopterin levels were observed in all groups compared with MPD. Increased hCat1 protein and L-arginine transport were observed in both GDM groups compared with MPD. However, the transport was higher in GDM-MSPD compared to GDM-MPD. Higher Arginase II protein and activity were observed in GDM-MSPD compared with MPD. Thus, MSPD in GDM pregnancies alters fetal endothelial function associated with NO metabolism.

Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.

Differentiation of methanosaeta concilii and methanosarcina barkeri in anaerobic mesophilic granular sludge by fluorescent In situ hybridization and confocal scanning laser microscopy

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

The interferon-inducible protein kinase PKR modulates the transcriptional activation of immunoglobulin kappa gene.

PKR is an interferon (IFN)-induced serine/threonine protein kinase that regulates protein synthesis through phosphorylation of eukaryotic translation initiation factor-2 (eIF-2). In addition to its demonstrated role in translational control, recent findings suggest that PKR plays an important role in regulation of gene transcription, as PKR phosphorylates I kappa B alpha upon double-stranded RNA treatment resulting in activation of NF-kappa B DNA binding in vitro (Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B.R.G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6288-6292). To further investigate the role of PKR in transcriptional signaling, we expressed the wild type human PKR and a catalytically inactive dominant negative PKR mutant in the murine pre-B lymphoma 70Z/3 cells. Here, we report that expression of wild type PKR had no effect on kappa-chain transcriptional activation induced by lipopolysaccharide or IFN-gamma. However, expression of the dominant negative PKR mutant inhibited kappa gene transcription independently of NF-kappa B activation. Phosphorylation of eIF-2 alpha was not increased by lipopolysaccharide or IFN-gamma, suggesting that PKR mediates kappa gene transcriptional activation without affecting protein synthesis. Our findings further support a transcriptional role for PKR and demonstrate that there are at least two distinct PKR-mediated signal transduction pathways to the transcriptional machinery depending on cell type and stimuli, NF-kappa B-dependent and NF-kappa B-independent.

HLA-DR polymorphism affects the interaction with CD4.

Major histocompatibility complex (MHC) class II molecules are highly polymorphic and bind peptides for presentation to CD4+ T cells. Functional and adhesion assays have shown that CD4 interacts with MHC class II molecules, leading to enhanced responses of CD4+ T cells after the activation of the CD4-associated tyrosine kinase p56lck. We have addressed the possible contribution of allelic polymorphism in the interaction between CD4 and MHC class II molecules. Using mouse DAP-3-transfected cells expressing different isotypes and allelic forms of the HLA-DR molecule, we have shown in a functional assay that a hierarchy exists in the ability of class II molecules to interact with CD4. Also, the study of DR4 subtypes minimized the potential contribution of polymorphic residues of the peptide-binding groove in the interaction with CD4. Chimeras between the DR4 or DR1 molecules, which interact efficiently with CD4, and DRw53, which interacts poorly, allowed the mapping of polymorphic residues between positions beta 180 and 189 that can exert a dramatic influence on the interaction with CD4.

Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands.

The binding of insulin to cultured IM-9 human lymphocytes was studied by flow cytometry using FITC-insulin and biotinylated insulins coupled to streptavidin-phycoerythrin (N alpha beta 1-biotinylinsulin (B-insulin) and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin (NBC-insulin)). The reference methods were 125I-insulin binding and the insulin-antiinsulin antibody complexes for flow cytometry. There was a close correlation between 125I-insulin binding and increase in fluorescence for B-insulin, NBC-insulin, and insulin-anti-insulin antibody complexes, but not for FITC-insulin. NBC-insulin gave the largest increase in fluorescence (79 +/- 9 channels) and the the insulin-antiinsulin antibody complexes the smallest (34 +/- 2 channels) (P < 0.05). FITC-insulin and B-insulin gave similar results: 47 +/- 6 and 59 +/- 6 channels. The concentration reducing 125I-insulin binding by 50% was 1.1 x 10(-9) M for native insulin, 2.7 x 10(-9) M for B-insulin, 3.3 x 10(-9) M for NBC-insulin, and 6.6 x 10(-9) M for FITC-insulin (P < 0.05). Nonspecific binding was low for B-insulin and NBC-insulin but reached 75% for 10(-6) M FITC-insulin. These results suggest that B-insulin and NBC-insulin are suitable ligands for insulin binding studies using flow cytometry. This two-step procedure is easier than the insulin-antiinsulin antibody complex technique. Its poor affinity, specificity, and sensitivity make FITC-insulin less suitable.

New chamber for flow cytometric analysis over an extended range of stream velocity and application to cell adhesion measurements.

When analyzed in a flow cytometer, particles are suddenly accelerated to high velocities (1-10 m.s-1) over very short distances. This feature is essential to obtain high analysis rates and low coincidence levels, but translates into very strong velocity gradients (greater than 10(5) s-1): particles experience strong hydrodynamic stresses that elongate them and tend to dissociate weakly associated complexes. In order to analyze fragile conjugates formed by heterotypic adhesion between two cell types, a flow cytometer was modified to make hydrodynamic stress not only much weaker but also adjustable. A new and easily adaptable flow cell was designed for the instruments of the FACS series; it provided satisfactory hydrodynamic conditions on a wide continuous range of flow rates. Accompanying electronic adaptations permitted standard analysis between 0.01 and 10 m.s-1. At 0.01 m.s-1, the velocity gradient roughly amounts to 50 s-1. Conjugates formed by the adhesion between human B and resting T lymphocytes, disrupted in conventional flow cytometers, could be detected and precisely quantified provided analysis velocity was kept below 0.1 m.s-1. We conclude that low velocity flow cytometry makes possible the quantification of weak intercellular adhesion phenomena, and is potentially useful for the future development of new biomechanical techniques and other applications.

Mutational analysis of the interaction between CD4 and class II MHC: class II antigens contact CD4 on a surface opposite the gp120-binding site.

Using functional and adhesion assays, we have studied the ability of 30 human CD4 mutants to interact with class II major histocompatibility complex (MHC) molecules and also with gp120 from human immunodeficiency virus. The mutants cover the four domains (D1-D4) of CD4 and include several single-site substitutions. Analysis of the results, in the context of the CD4 crystal structure, shows that mutations that affect the interaction with class II MHC molecules are located on three exposed loops from CD4 domains 1 and 2. The specifically implicated residues, 19, 89, and 165, are separated from one another by 9 A, 24 A, and 24 A on one face of the CD4 molecule. Moreover, the class II binding site does not include residues 43 to 49 of the CD4 molecule, a region on an opposite face known to be involved in the binding of gp120.

Developmental changes in the half-life of acetylcholine receptors in the myotomal muscle of Xenopus laevis.

1. Tail preparations, containing myotomal muscle and associated spinal cord, were isolated from embryos and tadpoles of Xenopus laevis between stages 25 and 49 (1.15-12 days) and were pulse-labelled with 125I-alpha-bungarotoxin (125I alpha BT) so that the half-life (T1/2) of their acetylcholine receptors (AChRs) could be estimated in organ culture. 2. For the entire population of AChRs, estimates of T1/2 based on a single exponential decline in radioactivity (but see item 4 below) increased from 53-55 h at stages 25-31 (1.15-1.56 days) to approximately 135 h at stage 47 (5.5 days). Beyond stage 47 T1/2 increased only slightly. 3. Radioautographic estimates of the T1/2 of extrajunctional AChRs at stages 47-48 (5.5-7.5 days) were 41-50 h. It follows that the developmental change in the T1/2 of the entire population of AChRs was due to the junctional AChRs. 4. At stages 47-49 (5.5-12 days) the decline in radioactivity for the entire population of AChRs was fitted well by a double exponential. Assuming a T1/2 of 50 h for the extrajunctional AChRs and 210 h for the junctional AChRs, the correlation coefficient (r) was 0.9947 +/- 0.0014 (mean +/- S.E.M.; n = 14) and junctional AChRs were estimated to comprise 80 +/- 3% of the entire population. Similar analysis, as well as experiments in which the degradation of junctional AChRs was assessed by pulse-labelling with fluorescent alpha-bungarotoxin, suggested that at earlier stages of development the junctional AChRs have a shorter T1/2 and comprise a smaller fraction of the entire population. 5. The developmental increase in T1/2 occurred even when animals were raised in the anaesthetic tricaine or in tetrodotoxin, conditions which abolished all motor activity. 6. Developmental increases in T1/2 also occurred in culture but were smaller than those in vivo. The increases in culture did not occur amongst those AChRs which were pre-labelled with 125I alpha BT. 7. It is concluded that in Xenopus myotomal muscle the T1/2 of junctional AChRs begins to increase within a day after the onset of innervation and that the increase does not require nerve or muscle impulse activity. We suggest, among other possibilities, that it may depend upon incorporation of a different molecular species of AChR into the postsynaptic membrane.

Intracellular and surface acetylcholine receptors during the normal development of a frog skeletal muscle.

125I-alpha-bungarotoxin (125I-alpha BT) was used to measure the pool sizes of surface and intracellular acetylcholine receptors (AChRs) in the myotomal muscle of Xenopus laevis over a developmental period (stages 23-48; 1.03-7.5 d) which ranged from initial to mature stages of neuromuscular synaptogenesis. The surface pool increased progressively throughout development. The intracellular pool increased more slowly and also underwent a transient decrease. Linear regression indicated that AChRs begin to appear intracellularly and in the surface membrane at embryonic ages of 13.2 and 18.5 hr, respectively. The findings also suggest that newly synthesized AChRs contribute much more to the intracellular pool than do AChRs internalized from the surface membrane and that the rates of supply and/or intracellular resident times of these 2 sources of intracellular AChRs change during the course of normal development. Carbamylcholine, even at concentrations 10-fold greater than needed to block completely 125I-alpha BT binding to surface AChRs, blocked specific intracellular binding by only 80%. Considered in the light of previous studies on cell cultures, these results suggest that 20% of the intracellular sites are on alpha-subunits not yet assembled into pentameric AChRs. Light microscope radioautography revealed an essentially uniform distribution of intracellular AChRs along the length of the muscle cells. It is concluded that during the normal development of Xenopus myotomal muscle the accumulation and maintenance of AChRs in the postsynaptic membrane occurs in the absence of any preferential concentration of intracellular AChRs in the subsynaptic region.