Kinetic and pharmacological analysis of L-[35S]cystine transport into rat brain synaptosomes

The synaptosomal transport of L-[35S]cystine occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and the specificity of inhibitors. They are (a) low affinity sodium-dependent transport (Km 463 +/- 86 microM, Vmax 185 +/- 20 nmol mg protein-1 min-1), (b) high affinity sodium-independent transport (Km 6.90 +/- 2.1 microM, Vmax 0.485 +/- 0.060 nmol mg protein(-1) min(-1)) and (c) low affinity sodium-independent transport (Km 327 +/- 29 microM, Vmax 4.18 +/- 0.25 nmol mg protein(-1) min(-1)). The sodium-dependent transport of L-cystine was mediated by the X(AG)- family of glutamate transporters, and accounted for almost 90% of the total quantity of L-[35S]cystine accumulated into synaptosomes. L-glutamate (Ki 11.2 +/- 1.3 microM) was a non-competitive inhibitor of this transporter, and at 100 microM L-glutamate, the Vmax for L-[35S]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[3H]aspartate into synaptosomes. L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[35S]cystine. L-homocysteate, L-cysteine sulphinate and L-homocysteine sulphinate were also effective inhibitors. 1 mM L-glutamate reduced the sodium-independent transport of L-cystine to 63% of control. These results suggest that the vast majority of the L-cystine transported into synaptosomes occurs by the high-affinity glutamate transporters, but that L-cystine may bind to a site that is distinct from that to which L-glutamate binds. The uptake of L-cystine by this mechanism is sensitive to inhibition by increased extracellular concentrations of L-glutamate. The importance of these results for understanding the mechanism of glutamate-mediated neurotoxicity is discussed.     

Correction to: Microhabitats of sharknose goby (Elacatinus evelynae) cleaning stations and their links with cleaning behaviour

Coral Reefs - A correction to this paper has been published: https://doi.org/10.1007/s00338-021-02111-z     

2G12-Expressing B Cell Lines May Aid in HIV Carbohydrate Vaccine Design Strategies

The highly conserved cluster of high-mannose glycans on the HIV-1 envelope glycoprotein, gp120, has been highlighted as a target for neutralizing antibodies. 2G12, the first HIV-1 antiglycan neutralizing antibody described, binds with an unusual domain-exchanged structure that creates a high-affinity multivalent binding surface. It is an interesting challenge for rational vaccine design to generate immunogens capable of eliciting domain-exchanged 2G12-like responses. We recently showed that di-mannose recognition by the variable domains of 2G12 is independent of domain exchange but that exchange is critical for virus neutralization. Carbohydrate-based immunogens aimed at inducing 2G12-like antibodies may need to drive both di-mannose recognition and domain exchange through interactions with B cell receptors. Here we assessed the ability of such immunogens to activate mouse B cell lines displaying domain-exchanged wild-type 2G12 (2G12 WT), a non-domain-exchanged Y-shaped variant (2G12 I19R), and germ line 2G12 (2G12 gl). We show that several immunogens, including heat-killed yeast and bacteria, can activate both 2G12 WT and 2G12 I19R B cells. However, only discrete clusters of high-mannose glycans, as on recombinant forms of the HIV-1 envelope trimer and oligodendrons, activate 2G12 WT B cells. Furthermore, no immunogen tested activated 2G12 gl cells. Our results support the hypothesis that in order to drive domain exchange of an antimannose antibody response, a boost with an immunogen displaying discrete clusters of high-mannose glycans not recognized by conventional Y-shaped antibodies will be required. Additionally, a molecule capable of activating 2G12 gl cells might also be required. The results highlight broadly neutralizing antibody-expressing mouse B cells as potentially useful tools for carbohydrate immunogen screening.     

Identification of blood-protein carriers of the CA 19-9 antigen and characterization of prevalence in pancreatic diseases

The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using MS. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10-25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.     

Differential Effector Engagement by Oncogenic KRAS

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AICAR induces phosphorylation of AMPK in an ATM-dependent, LKB1-independent manner

AMPK is an AMP-activated protein kinase that plays an important role in regulating cellular energy homeostasis. Metabolic stress, such as heat shock and glucose starvation, causes an energy deficiency in the cell and leads to elevated levels of intracellular AMP. This results in the phosphorylation and activation of AMPK. LKB1, a tumor suppressor, has been identified as an upstream kinase of AMPK. We found that in response to treatment with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), the LKB1 deficient cancer cell line, HeLa, exhibited AMPK-α phosphorylation. This indicates the existence of an LKB1-independent AMPK-α phosphorylation pathway. ATM is a protein that is deficient in the disease ataxia telangiectasia (A-T). We measured the activation of AMPK by AICAR in the normal mouse embryo fibroblast cell line, A29, and the mouse cell line lacking the ATM protein, A38. In A38 cells, the level of AICAR-induced AMPK-α phosphorylation was significantly lower than that found in A29 cells. Furthermore, phosphorylation of AMPK in HeLa and A29 cells was inhibited by an ATM specific inhibitor, KU-55933. Our results demonstrate that AICAR treatment could lead to phosphorylation of AMPK in an ATM-dependent and LKB1-independent manner. Thus, ATM may function as a potential AMPK kinase in response to AICAR treatment.     

Observation of sequence specificity in the seeding of protein amyloid fibrils

It is well established that the rate of formation of fibrils by amyloidogenic proteins is enhanced by the addition of preformed fibrils, a phenomenon known as seeding. We show that the efficiency of seeding fibril formation from solutions of hen lysozyme by a series of other proteins depends strongly on the similarity of their sequences. This observation is consistent with the importance of long-range interactions in stabilizing the core structure of amyloid fibrils and may be associated with the existence of a species barrier observed in the transmissible spongiform encephalopathies. In addition, it is consistent with the observation of a single dominant type of protein in the deposits associated with each form of amyloid disease.     

Global DNA methylation (LINE-1) associated with exposure to arsenic-contaminated environment and with type of arsenical skin lesions in Thailand

The effects of chronic arsenic exposure mode on DNA methylation and skin lesion type are unclear. These relationships were investigated in an arsenic-contaminated area of southern Thailand. Cases with arsenical skin lesions (n = 131) and lesion-free controls (n = 163) were selected from an arsenic-contaminated sub-district, as well as 105 controls from a non-contaminated area. Type and severity of skin lesions and salivary global DNA methylation (LINE-1) were determined. Arsenic exposure was characterized as occupational, domestic and current (toe-nail arsenic). Associations were explored using logistic regression. Cases and controls had lower LINE-1 methylation and higher toenail arsenic than external controls (74.65% and 74.61% vs 76.05%, p < 0.001 for each). Cases were more likely to have been exposed domestically (OR_total 1.76, 95% ci 1.00, 3.11; and 2.22, 95% ci 1.22, 4.03; P_trend = 0.005 for exposure <36 and ≥36 years). More severe spotty hyperpigmentation was related to higher LINE-1 methylation (P_trend=0.006). LINE-1 methylation was positively associated with toenail arsenic only among non-symptomatic exposed subjects (OR 1.31, 95% ci 1.06, 1.64; p = 0.014). Exposure to an arsenic-contaminated environment results in global DNA hypomethylation. However, among symptomatic subjects, increased global DNA methylation was associated with increased severity of spotty hyperpigmentation.