Identification of autoantibodies against TRPM1 in patients with paraneoplastic retinopathy associated with ON bipolar cell dysfunction

Background

Paraneoplastic retinopathy (PR), including cancer-associated retinopathy (CAR) and melanoma-associated retinopathy (MAR), is a progressive retinal disease caused by antibodies generated against neoplasms not associated with the eye. While several autoantibodies against retinal antigens have been identified, there has been no known autoantibody reacting specifically against bipolar cell antigens in the sera of patients with PR. We previously reported that the transient receptor potential cation channel, subfamily M, member 1 (TRPM1) is specifically expressed in retinal ON bipolar cells and functions as a component of ON bipolar cell transduction channels. In addition, this and other groups have reported that human TRPM1 mutations are associated with the complete form of congenital stationary night blindness. The purpose of the current study is to investigate whether there are autoantibodies against TRPM1 in the sera of PR patients exhibiting ON bipolar cell dysfunction.

Methodology/Principal Findings

We performed Western blot analysis to identify an autoantibody against TRPM1 in the serum of a patient with lung CAR. The electroretinograms of this patient showed a severely reduced ON response with normal OFF response, indicating that the defect is in the signal transmission between photoreceptors and ON bipolar cells. We also investigated the sera of 26 patients with MAR for autoantibodies against TRPM1 because MAR patients are known to exhibit retinal ON bipolar cell dysfunction. Two of the patients were found to have autoantibodies against TRPM1 in their sera.

Conclusion/Significance

Our study reveals TRPM1 to be one of the autoantigens targeted by autoantibodies in at least some patients with CAR or MAR associated with retinal ON bipolar cell dysfunction.     

Stimulation of DNA synthesis by heated human serum in primary cultured normal adult rat hepatocytes

In primary culture of normal adult rat hepatocytes, human serum heated at 56°C for 30 min stimulated dose-dependently [³H]thymidine incorporation into trichloroacetic acid insoluble fraction of the cells, most of which was solubilized into hot trichloroacetic acid solution. The solubilized fraction was reduced when hydroxyurea was added to the culture. The heated serum also increased dose-dependently protein synthesis and cell viability determined from morphological findings. These results suggest that human serum has heat-stable factors stimulating DNA synthesis and maintaining cell viability of cultured rat hepatocytes.     

Quantitative contribution of the acid production to the intracellular acidification in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O₂ ^–) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O₂ ^–, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/10⁶ cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO₂ in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/10⁶ cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.Abbreviations fMLP N-formyl-methionyl-leucyl-phenylalanine - BCECF-AM 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester - PMA phorbol 12-myristate 13-acetate - CGD chronic granulomatous disease - HMP hexose monophosphate - pHi intracellular pH     

Reconstitution of bacteriorhodopsin into cyclic lipid vesicles

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.     

IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Quantification of Cellular NEMO Content and Its Impact on NF-κB Activation by Genotoxic Stress

NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 10⁵ molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6–6x10⁵ molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.     

Gender difference in N170 elicited under oddball task

Background

Some studies have reported gender differences in N170, a face-selective event-related potential (ERP) component. This study investigated gender differences in N170 elicited under oddball paradigm in order to clarify the effect of task demand on gender differences in early facial processing.

Findings

Twelve males and 10 females discriminated targets (emotional faces) from non-targets (emotionally neutral faces) under an oddball paradigm, pressing a button as quickly as possible in response to the target. Clear N170 was elicited in response to target and non-target stimuli in both males and females. However, females showed more negative amplitude of N170 in response to target compared with non-target, while males did not show different N170 responses between target and non-target.

Conclusions

The present results suggest that females have a characteristic of allocating attention at an early stage when responding to faces actively (target) compared to viewing faces passively (non-target). This supports previous findings suggesting that task demand is an important factor in gender differences in N170.     

Inter-individual relationships in empathic traits and feedback-related fronto-central brain activity: an event-related potential study

Background

Neuroimaging studies continue to indicate the major role the anterior cingulate cortex (ACC) plays in processing empathic responses. Error-related negativity (ERN), an event-related potential (ERP) thought to arise from the ACC, has been found to correlate with scores for individual empathic personality. This study investigated the relationship between empathic personality traits and the amplitude of feedback-related negativity (FRN), an ERP sourced from the ACC and similar to the ERN, using a task involving feedback of monetary gains or losses.

Methods

Sixteen healthy participants answered an empathy trait questionnaire and performed a gambling task to elicit FRN. Because FRN amplitude is thought to be associated with attention, motivation, emotional state, and anxiety trait, we performed a partial correlation analysis between the empathic trait score and FRN amplitude while controlling for variables.

Results

In partial correlation analysis, FRN amplitude was significantly inversely correlated with scores for personal distress and marginally correlated with scores for empathic concern and with total average score.

Discussion

The study revealed for the first time an association between FRN and emotional empathic traits, after controlling for variables that can affect FRN amplitude. However, we also found a reversed directional correlation contrary to our expectations. This fronto-central brain activity may be associated with empathic properties via dopaminergic neuronal function. Future study using these electric potentials as experimental tools is expected to help elucidate the neurological mechanism of empathy.