Change of mouse CD5(+) B1 cells to a macrophage-like morphology induced by gamma interferon and inhibited by interleukin-4

The in vitro effects of gamma interferon (IFN-gamma) on the mouse CD5(+) B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-gamma, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-gamma. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-gamma-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-gamma and IL-4 exhibited contradictory actions on morphological change of CD5(+) B1 cells into macrophage-like cells. Differential regulation of CD5(+) B1 cells by IFN-gamma, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.     

Comparative studies on the actions of antigen and polyclonal B-cell activator in differentiation and proliferation of B-cells and B memory cells.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T cell-dependent antigen, we compared the ability of PBA and antigen to differentiate (generate antibody-forming cells, AFC) and proliferate (generate immunological memory) virgin B cells and B memory cells. In vitro CPS-K induced the differentiation of IgM virgin B cells, IgM B memory cells and IgG B memory cells to AFC, as well as or better than SRBC. The differentiation of B memory cells to AFC by CPS-K did not require the participation of macrophages or T cells, whereas the action of SRBC depended strictly upon the helper actions of these cells. The responsiveness to CPS-K and SRBC of normal and antigen-primed spleen cells as judged by anti-SRBC PFC responses in vitro was markedly decreased after stimulation of virgin B cells and B memory cells in vivo by CPS-K injection into normal or primed mice but greatly increased after the injection of SRBC. The decrease in the responsiveness to CPS-K of spleen cells from mice treated with CPS-K appeared principally due to exhaustion of the functions of B cells and B memory cells. From the present data it has been concluded that the signals required for the differentiation and proliferation of B cells of B memory cells are different from each other, the signal for differentiation being provided by either antigen (SRBC) or PBA (CPS-K), while the signal for proliferation only by antigen.     

Recurrent cerebral infarctions and del(10)(p14p15.1) de novo in HDR (hypoparathyroidism,sensorineural deafness,renal dysplasia) syndrome

We report on a Japanese boy with HDR syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia) and recurrent cerebral infarctions in the basal ganglia. The patient experienced cerebral infarctions four times between age 7 months and age 20 months. Chromosome analysis of the patient demonstrated a 46,XY,del(10)(p14p15.1) de novo. This suggests that the putative gene responsible for HDR syndrome is located at 10p14-p15.1. Am. J. Med. Genet. 86:427–429, 1999. © 1999 Wiley-Liss, Inc.     

Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.

Plant chloroplasts have evolved multiple strategies with which to adapt photosynthesis to fluctuating light environments. One such strategy involves the redox regulation of various enzymes that function in photosynthesis reactions. Multiple photosynthesis-related proteins, such as the four Calvin–Benson cycle enzymes (glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase [FBPase], sedoheptulose-1,7-bisphosphatase [SBPase], and phosphoribulokinase [PRK]), possess redox-active Cys residues (1, 2). In addition, the γ-subunit of ATP synthase (CF₁-γ) and two regulatory proteins associated with Calvin–Benson cycle enzymes, CP12 and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA), are also redox-regulated (2–4). In the 1970s thioredoxin (Trx) was identified as a reducing power mediator for FBPase and SBPase in chloroplasts (5, 6). In a light-containing environment, reducing power is transferred from the photosynthetic electron transport system to Trx via ferredoxin and ferredoxin-Trx reductase (6). Trx then achieves light-dependent activation of its target enzymes by reducing the disulfide bond on these enzymes.In chloroplasts, NADPH-Trx reductase C (NTRC) works in parallel with the Trx-dependent system as another redox pathway. NTRC is also a redox-responsive protein containing both NADPH-dependent Trx reductase and Trx domains; these enable NTRC to reduce its target proteins using the reducing power of NADPH (7). NTRC can reduce 2-Cys peroxiredoxin (2-Cys Prx) in addition to several Trx-targeted proteins (8–12). 2-Cys Prx utilizes reducing power to reduce reactive oxygen species such as H₂O₂ (13). In chloroplasts, NTRC is thought to be a major electron donor for 2-Cys Prx (14) because the reducing power transfer efficiency from NTRC is extremely high compared with that from typical chloroplast Trx proteins (12). Plants deficient in NTRC show severe phenotypes, such as stunted growth, low chlorophyll content, and very high nonphotochemical quenching (NPQ) (7, 11, 12, 14–17). Thus, it is clear that NTRC plays important physiological roles in chloroplasts.Redox-regulated proteins in the stroma are reduced in the light and then reoxidized in the dark (18, 19). Reoxidation is an important process in plants; for example, we recently showed that the reoxidation of chloroplast NADP-malate dehydrogenase is important for maintaining NADPH homeostasis in chloroplasts, particularly in an environment with fluctuating light (20). Despite the importance of the oxidation process, the proteins involved in target oxidation have yet to be clarified. Recently, Trx-like proteins, such as Trx-like 2 (TrxL2) and atypical Cys His-rich Trx (ACHT), have been suggested as oxidation factors (21–27). These reports were mainly based on the results of in vitro experiments, suggesting that Trx-like proteins transfer the reducing power of Trx-targeted proteins to H₂O₂ via 2-Cys Prx. However, the functions of these proteins in vivo are not known very well. The so-called common Trxs belonging to f-, m-, x-, y- (or z-?) types were also thought to be the candidate of the oxidation factor. Because it is known that, particularly, Trx-f can oxidize its target proteins under certain conditions in vitro (25, 28), we focused this work on Trx-f.Target oxidation by ACHT1 and ACHT2, among five ACHT isoforms in Arabidopsis thaliana (29), has been demonstrated in vitro (25). ACHT1 and ACHT2 are broadly conserved in photosynthetic organisms, including green algae, moss, and seed plants (30). Their amino acid sequences and biochemical properties are similar (SI Appendix, Fig. S1A) (25, 29). In addition, ACHT1 and ACHT2 (designated also as Lilium5 and Lilium2, respectively) are predicted to originate from the same ancestral gene (31). Comparison of the expression patterns of ACHT1 and ACHT2 in the database shows that ACHT2 is expressed more than ACHT1, especially in leaves (SI Appendix, Fig. S1B) (32), suggesting that ACHT2 may play a dominant role in A. thaliana leaves.Oxidation of target proteins by the TrxL2 isoforms from A. thaliana, namely TrxL2.1 and TrxL2.2, has been demonstrated in vitro (22). TrxL2 genes are also conserved in photosynthetic organisms, such as seed plants, moss, and some green algae, but not in Chlamydomonas reinhardtii (30). Although the amino acid sequences and biochemical properties of TrxL2.1 and TrxL2.2 are similar (SI Appendix, Fig. S2A) (22), their expression patterns are different, and TrxL2.1 is reported to be more expressed than TrxL2.2, particularly in leaves (SI Appendix, Fig. S2B) (32). In addition, TrxL2.1 expression seems to be regulated by the circadian rhythm and the rhythm of temperature change (SI Appendix, Fig. S2C). The expression of TrxL2.1 is more strongly induced before and during light-to-dark transitions than TrxL2.2, suggesting that TrxL2.1 plays a predominant role during these periods.In the present study, we generated A. thaliana mutant plant lines deficient in Trx-f1 and Trx-f2, TrxL2.1, or ACHT1 and ACHT2, whose target oxidation activities are well studied in vitro, and used these plants to investigate redox state changes in chloroplasts. We found that Trx-f, TrxL2.1, ACHT1, and ACHT2 are involved in the oxidation of FBPase, CF₁-γ, and RCA. ACHT2 also seemed to be involved in the regulation of NPQ. Furthermore, the knockout of Trx-like proteins suppressed the impact of NTRC deficiency in plants, suggesting that a connection existed between the NTRC system and Trx-like protein-involving system.     

Functional analysis of LAT3 in prostate cancer: Its downstream target and relationship with androgen receptor

L-type amino acid transporter 3 (LAT3, SLC43A1) is abundantly expressed in prostate cancer (PC) and is thought to play an essential role in PC progression through the cellular uptake of essential amino acids. Here, we analyzed the expression, function, and downstream target of LAT3 in PC. LAT3 was highly expressed in PC cells expressing androgen receptor (AR), and its expression was increased by dihydrotestosterone treatment and decreased by bicalutamide treatment. In chromatin immunoprecipitation sequencing of AR, binding of AR to the SLC43A1 region was increased by dihydrotestosterone stimulation. Knockdown of LAT3 inhibited cell proliferation, migration, and invasion, and the phosphorylation of p70S6K and 4EBP-1. Separase (ESPL1) was identified as a downstream target of LAT3 by RNA sequencing analysis. In addition, immunostaining of prostatectomy specimens was performed. In the multivariate analysis, high expression of LAT3 was an independent prognostic factor for recurrence-free survival (hazard ratio: 3.24; P = .0018). High LAT3 expression was correlated with the pathological T stage and a high International Society of Urological Pathology grade. In summary, our results suggest that LAT3 plays an important role in the progression of PC.     

Novel pathogenic mutations and copy number variations in the VPS13A Gene in patients with chorea‐acanthocytosis

Chorea‐acanthocytosis (ChAc) is a rare autosomal recessive neurodegenerative disorder caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene that encodes chorein. It is characterized by adult‐onset chorea, peripheral acanthocytes, and neuropsychiatric symptoms. In the present study, we performed a comprehensive mutation screen, including sequencing and copy number variation (CNV) analysis, of the VPS13A gene in ChAc patients. All 73 exons and flanking regions of VPS13A were sequenced in 35 patients diagnosed with ChAc. To detect CNVs, we also performed real‐time quantitative PCR and long‐range PCR analyses for the VPS13A gene on patients in whom only a single heterozygous mutation was detected. We identified 36 pathogenic mutations, 20 of which were previously unreported, including two novel CNVs. In addition, we investigated the expression of chorein in 16 patients by Western blotting of erythrocyte ghosts. This demonstrated the complete absence of chorein in patients with pathogenic mutations. This comprehensive screen provides an accurate and useful method for the molecular diagnosis of ChAc. © 2011 Wiley‐Liss, Inc.     

Spinal segmental myoclonus during postural maintenance in a patient with cervical spondylosis: a case report

Spinal segmental myoclonus is defined as a rare involuntary movement characterized by myoclonic jerks of spinal origin. We describe the case of a 62-year-old woman who developed spinal segmental myoclonus 4 months after undergoing cervical laminoplasty for ossification of the posterior longitudinal ligament. Myoclonic jerks were observed in the upper trapezius innervated by C3-4, which corresponded to the level of myelomalacia. These jerks were elicited and aggravated in the sitting and standing positions but were completely suppressed in the supine position. The myoclonus was refractory to medication but improved with the use of a soft neck brace.     

Association of nodular hyperplasia with resistance to cinacalcet therapy for secondary hyperparathyroidism in hemodialysis patients

There have been few long-term prospective studies investigating the effect of cinacalcet on secondary hyperparathyroidism with or without nodular hyperplasia. We examined whether the effect of cinacalcet on secondary hyperparathyroidism differed between patients with or without nodular hyperplasia. Stable hemodialysis patients with secondary hyperparathyroidism resistant to conventional treatment received cinacalcet for 12 months. Based on ultrasonography findings, patients were divided into group S (gland < 500 mm(3) without nodular hyperplasia) and group L (gland ≥ 500 mm(3) with nodular hyperplasia). Serum levels of intact parathyroid hormone, bone-specific alkaline phosphatase, osteocalcin, and cross-linked N-terminal telopeptide of type 1 collagen were measured. Thirty-one patients completed the study. The changes of parameters from the baseline did not differ significantly between the two groups after 6 months. However, the percentage reduction of each parameter was significantly smaller in group L compared with group S after 12 months. Nodular hyperplasia is associated with resistance to cinacalcet therapy in patients on chronic dialysis with secondary hyperparathyroidism.     

Scent test using Caenorhabditis elegans to screen for early-stage pancreatic cancer

Although early detection and diagnosis are indispensable for improving the prognosis of patients with pancreatic cancer, both have yet to be achieved. Except for pancreatic cancer, other cancers have already been screened through scent tests using animals or microorganisms, including Caenorhabditis elegans. While such a method may greatly improve the prognosis of pancreatic cancer, no studies have investigated the same, mainly given the difficulty of collecting suitable samples from patients with early-stage pancreatic cancer. In this study, we organized a nationwide study group comprising high-volume centers throughout Japan to collect patients with very-early-stage pancreatic cancer (stage 0 or IA). We initially performed an open-label study involving 83 cases (stage 0–IV), with subsequent results showing significant differences after surgical removal in stage 0–IA (×10 dilution: p < 0.001; ×100 dilution: p < 0.001). Thereafter, a blinded study on 28 cases (11 patients with stage 0 or IA disease and 17 healthy volunteers) was conducted by comparing very-early-stage pancreatic cancer patients with healthy volunteers to determine whether C. elegans could detect the scent of cancer for the diagnosis of early-stage pancreatic cancer. Preoperative urine samples had a significantly higher chemotaxis index compared to postoperative samples in patients with pancreatic cancer [×10 dilution: p < 0.001, area under the receiver operating characteristic curve (AUC) = 0.845; ×100 dilution: p < 0.001, AUC = 0.820] and healthy volunteers (×10 dilution: p = 0.034; ×100 dilution: p = 0.088). Moreover, using the changes in preoperative and postoperative chemotaxis index, this method had a higher sensitivity for detecting early pancreatic cancer compared to existing diagnostic markers. The clinical application C. elegans for the early diagnosis of cancer can certainly be expected in the near future.