Lysophosphatidic acid induces endothelial cell death by modulating the redox environment

Oxidant stress plays a significant role in hypoxic-ischemic injury to the susceptible microvascular endothelial cells. During oxidant stress, lysophosphatidic acid (LPA) concentrations increase. We explored whether LPA caused cytotoxicity to neuromicrovascular cells and the potential mechanisms thereof. LPA caused a dose-dependent death of porcine cerebral microvascular as well as human umbilical vein endothelial cells; cell death appeared oncotic rather than apoptotic. LPA-induced cell death was mediated via LPA(1) receptor, because the specific LPA(1) receptor antagonist THG1603 fully abrogated LPA's effects. LPA decreased intracellular GSH levels and induced a p38 MAPK/JNK-dependent inducible nitric oxide synthase (NOS) expression. Pretreatment with the antioxidant GSH precursor N-acetyl-cysteine (NAC), as well as with inhibitors of NOS [N(omega)-nitro-l-arginine (l-NNA); 1400W], significantly prevented LPA-induced endothelial cell death (in vitro) to comparable extents; as expected, p38 MAPK (SB203580) and JNK (SP-600125) inhibitors also diminished cell death. LPA did not increase indexes of oxidation (isoprostanes, hydroperoxides, and protein nitration) but did augment protein nitrosylation. Endothelial cytotoxicity by LPA in vitro was reproduced ex vivo in brain and in vivo in retina; THG1603, NAC, l-NNA, and combined SB-203580 and SP600125 prevented the microvascular rarefaction. Data implicate novel properties for LPA as a modulator of the cell redox environment, which partakes in endothelial cell death and ensued neuromicrovascular rarefaction.     

Analysis of SRC Oncogenic Signaling in Colorectal Cancer by Stable Isotope Labeling with Heavy Amino Acids in Mouse Xenografts

The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [¹³C₆]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo.The non-receptor tyrosine kinase (TK)¹ SRC mediates cellular signaling induced by growth factors and integrins (1) leading to cell growth, survival, and migration. It also has oncogenic activity when deregulated, a role originally described for the constitutively active v-SRC (2) that has since been observed in a large variety of human cancers (3). Remarkably, elevated SRC kinase activity has been found in more than 80% of colorectal cancers (CRCs) to levels (5- to 10-fold) consistent with oncogenic properties (4). Moreover, SRC deregulation has been associated with poor clinical outcomes (3), suggesting an additional function of SRC during late tumorigenesis. SRC deregulation largely occurs in the absence of mutations in the SRC gene. Instead, it primarily involves protein over-expression (2) and inhibition of SRC negative regulators, such as the transmembrane protein Cbp/PAG (5, 6). A large body of evidence indicates that SRC deregulation is an important event in colon tumorigenesis (3, 6). Indeed, SRC controls growth, survival, and invasion of some CRC cell lines in vitro (4). Moreover, it contributes to tumor growth, angiogenesis, and metastasis formation in mouse colon tumor xenograft models (7–11). However, our knowledge of the SRC-dependent oncogenic signaling pathway in CRC is largely incomplete, mostly because the majority of data have been obtained in two-dimensional cell culture models. Moreover, the standard culture conditions of CRC cells do not allow the recapitulation of all the SRC-dependent signaling cascades that are activated during tumorigenesis to promote tumor growth, angiogenesis, and interactions with the microenvironment.MS-based quantitative phosphoproteomic technology has been a valuable tool for deciphering signaling pathways initiated by a given TK (12). Particularly, the method of stable isotope labeling with amino acids in cell culture (SILAC) has been employed for the characterization of oncogenic TK signaling pathways in cell culture, including HER2 (13) and BCR-ABL (14). We recently used this powerful approach to investigate SRC-dependent oncogenic signaling in CRC cells (15) and identified the first SRC-dependent tyrosine “phosphoproteome” in these cells. Additionally, we found that SRC phosphorylated a small cluster of TKs that mediate its oncogenic activity, thus uncovering a TK network that is important for the induction of CRC cell growth (15). Whether these signaling processes also operate in vivo is, however, currently unknown.SRC oncogenic signaling could be investigated in vivo using similar MS-based quantitative phosphoproteomic approaches in animal models or tumor biopsies. However, the application of the SILAC method in vivo has been challenging until recently because it requires efficient protein labeling in different tissues, which is conditioned by the rate of de novo protein synthesis. Recently, Mann et al. described the successful development of a SILAC approach for labeling mice that is based on the addition of [¹³C₆]-lysine to their food (16). They reported complete labeling from the F2 generation. Similar SILAC approaches were then described for additional multicellular organisms, such as worms (17), flies (18), and zebrafish (19). Here, we report a similar SILAC approach in which we labeled tumors in nude mice xenografted with human CRC cells. We reasoned that the high rate of de novo protein synthesis occurring in tumors should allow efficient tumor labeling in a short period of time. Indeed, we obtained consistent (>88%) labeling of the tumor proteome by feeding xenografted mice with the SILAC mouse diet for only 30 days. We then used this approach to compare the tyrosine phosphoproteome of SRC over-expressing tumors (labeled with heavy amino acids) and of control tumors (labeled with light amino acids) and report the first SRC-dependent tyrosine phosphoproteome of CRC in vivo. Finally, comparison of the in vivo and in vitro SRC-dependent tyrosine phosphoproteomes showed that some of the SRC substrates were specifically activated only in CRC xenograft tumors, and not in cultured CRC cells.     

Obesity and body fat classification in the metabolic syndrome: Impact on cardiometabolic risk metabotype

Objective:

Obesity is a key factor in the development of the metabolic syndrome (MetS), which is associated with increased cardiometabolic risk. We investigated whether obesity classification by BMI and body fat percentage (BF%) influences cardiometabolic profile and dietary responsiveness in 486 MetS subjects (LIPGENE dietary intervention study).

Design and Methods:

Anthropometric measures, markers of inflammation and glucose metabolism, lipid profiles, adhesion molecules, and hemostatic factors were determined at baseline and after 12 weeks of four dietary interventions (high saturated fat (SFA), high monounsaturated fat (MUFA), and two low fat high complex carbohydrate (LFHCC) diets, one supplemented with long chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFAs)).

Results:

About 39 and 87% of subjects classified as normal and overweight by BMI were obese according to their BF%. Individuals classified as obese by BMI (≥30 kg/m²) and BF% (≥25% (men) and ≥35% (women)) (OO, n = 284) had larger waist and hip measurements, higher BMI and were heavier (P < 0.001) than those classified as nonobese by BMI but obese by BF% (NOO, n = 92). OO individuals displayed a more proinflammatory (higher C reactive protein (CRP) and leptin), prothrombotic (higher plasminogen activator inhibitor‐1 (PAI‐1)), proatherogenic (higher leptin/adiponectin ratio) and more insulin resistant (higher HOMA‐IR) metabolic profile relative to the NOO group (P < 0.001). Interestingly, tumor necrosis factor‐α (TNF‐α) concentrations were lower post‐intervention in NOO individuals compared with OO subjects (P < 0.001).

Conclusions:

In conclusion, assessing BF% and BMI as part of a metabotype may help to identify individuals at greater cardiometabolic risk than BMI alone.     

Cell junctions acting as intestinal valves in nematodes

Serial sections of the rectal valve in Aphelenchoides blastophthorus Franklin and the oesophago-intestinal valve in Thornenema wickeni Yeates were examined electron microscopically. Each valve when closed appears as a convoluted path of closely apposed (10 nm) pairs of three-layered cell membranes. Both valves serve to stop intestinal leakage, open briefly and rapidly by forcible dilatation and are closed by pressure from surrounding tissues, helped perhaps by intermolecular forces.     

Phenylketonuria—Experience at One Center in the First Year of Screening in California

One year''s experience with phenylketonuria during the calendar year 1966, the first year for compulsory newborn screening in California, was reviewed. The over-all prevalence rate from reported cases in California during this period was one case per 19,500 persons tested. Fifty-seven persons suspected of having pku were evaluated, and 25 of them were determined to be phenylketonuric. Eleven of the 25 were infants in whom the abnormality was detected through the newborn screening program or because it was detected in a sibling through a screening program. All the newborn phenylketonuric patients were developing normally at the time of last report (although the follow-up periods were short).In nine of the other children, pku was detected because they were retarded. Five retarded children who were diagnosed as phenylketonuric at another clinic were given dietary assistance.Five additional infants had elevated serum phenylalanines but did not have the classic biochemical findings of pku and are being evaluated further. Nine infants with positive screening tests exhibited biochemical and clinical findings consistent with transient tyrosinemia. Eighteen other children were evaluated and found to have no metabolic abnormality.The newborn screening program for pku is of decided benefit in early identification of a group of infants who have a high rate of potentially serious metabolic disease. Early identification permits treatment soon enough to prevent mental retardation. Newly identified patients should be evaluated in a medical setting capable of careful pediatric, biochemical and nutritional surveillance.     

Correlates of preserved CD4(+) T cell homeostasis during natural, nonpathogenic simian immunodeficiency virus infection of sooty mangabeys: implications for AIDS pathogenesis

In contrast to HIV-infected humans, naturally SIV-infected sooty mangabeys (SMs) very rarely progress to AIDS. Although the mechanisms underlying this disease resistance are unknown, a consistent feature of natural SIV infection is the absence of the generalized immune activation associated with HIV infection. To define the correlates of preserved CD4(+) T cell counts in SMs, we conducted a cross-sectional immunological study of 110 naturally SIV-infected SMs. The nonpathogenic nature of the infection was confirmed by an average CD4(+) T cell count of 1,076 +/- 589/mm(3) despite chronic infection with a highly replicating virus. No correlation was found between CD4(+) T cell counts and either age (used as a surrogate marker for length of infection) or viremia. The strongest correlates of preserved CD4(+) T cell counts were a low percentage of circulating effector T cells (CD28(-)CD95(+) and/or IL-7R/CD127(-)) and a high percentage of CD4(+)CD25(+) T cells. These findings support the hypothesis that the level of immune activation is a key determinant of CD4(+) T cell counts in SIV-infected SMs. Interestingly, we identified 14 animals with CD4(+) T cell counts of <500/mm(3), of which two show severe and persistent CD4(+) T cell depletion (<50/mm(3)). Thus, significant CD4(+) T cell depletion does occasionally follow SIV infection of SMs even in the context of generally low levels of immune activation, lending support to the hypothesis of multifactorial control of CD4(+) T cell homeostasis in this model of infection. The absence of AIDS in these "CD4(low)" naturally SIV-infected SMs defines a protective role of the reduced immune activation even in the context of a significant CD4(+) T cell depletion.