Serum melatonin levels and insomnia in patients with Machado-Joseph Disease

We previously reported a patient with Machado-Joseph Disease (MJD) who had severe insomnia and a low serum melatonin (MLT) level, and whose insomnia was alleviated by oral MLT replacement therapy. The aims of this study were to examine whether patients with MJD are likely to have insomnia, and whether there is a relationship between the degree of insomnia and the serum MLT level among patients with MJD. This study included 8 patients with MJD. A 58-year-old-patient with cervical spondylosis was also included in this study to check the condition of the test room for sleeping. All patients filled out the Japanese version of Pittsburgh Sleep Quality Index (PSQI-J) questionnaire. We obtained blood samples at 12:00 and 24:00 hours to measure the MLT level. We checked the sleep condition of the patient once an hour and recorded the grade in sleep-logs: the grades of sleep condition were asleep, sleepy, or awake. Statistical analyses were performed to search for correlations between the PSQI score and the serum MLT level or actual sleep time using Spearman’s rank correlation coefficient. Seven of the 8 MJD patients had a total PSQI score of above 5.5 (cut-off level). The daytime MLT level (at 12:00 hours) was below 2.8 pg/mL in all 8 patients, whereas the mean night-time MLT level (at 24:00 hours) of the MJD patients (23.6 ± 17.5 pg/mL) was lower than that of the control patient (43.0 pg/mL) and also lower than the reported cut-off level among healthy people aged 30–50 years (55.5 pg/mL). There was a negative correlation between the total PSQI score and the serum MLT level among the MJD patients (P < 0.05). Our results show that a low serum MLT level may contribute to insomnia in patients with MJD.

     

Water-soluble fractions from defatted sesame seeds protect human neuroblast cells against peroxyl radicals and hydrogen peroxide-induced oxidative stress

Oxidative stress is involved in the development of aging-related diseases, such as neurodegenerative diseases. Dietary antioxidants that can protect neuronal cells from oxidative damage play an important role in preventing such diseases. Previously, we reported that water-soluble fractions purified from defatted sesame seed flour exhibit good antioxidant activity in vitro. In the present study, we investigated the protective effects of white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively) against 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H₂O₂) induced oxidative stress in human neuroblast SH-SY5Y cells. Pretreatment with WS-wsf and GS-wsf did not protect cells against AAPH-induced cytotoxicity, while simultaneous co-treatment with AAPH significantly improved cell viability and inhibited membrane lipid peroxidation. These results suggest that WS-wsf and GS-wsf protect cells from AAPH-induced extracellular oxidative damage via direct scavenging of peroxyl radicals. When oxidative stress was induced by H₂O₂, pretreatment WS-wsf and GS-wsf significantly enhanced cell viability. These results suggest that in addition to radical scavenging, WS-wsf and GS-wsf enhance cellular resistance to intracellular oxidative stress by activation of the Nrf-2/ARE pathway as confirmed by the increased Nrf2 protein level in the nucleus and increased heme oxygenase 1 (HO-1) mRNA expression. The roles of ferulic and vanillic acids as bioactive antioxidants in these fractions were also confirmed. In conclusion, our results indicated that WS-wsf and GS-wsf, which showed antioxidant activity in vitro, are also efficient antioxidants in a cell system protecting SH-SY5Y cells against both extracellular and intracellular oxidative stress.     

Identification of cis-localization elements of the maize 10-kDa δ-zein and their use in targeting RNAs to specific cortical endoplasmic reticulum subdomains

The RNAs for the storage proteins of rice ( Oryza sativa ), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis -localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis -localization elements (zip codes) at the 3' end of the RNA, whereas prolamine has two cis -elements; one located in the 5' end of the coding sequence and a second residing in the 3'-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins ('prolamine' class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa δ-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis -localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis -localization elements were identified; three in the coding sequences and one in the 3'-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species.     

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.     

cbb3-Type Cytochrome c Oxidases,Aerobic Respiratory Enzymes,Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1

For bacteria, many studies have focused on the role of respiratory enzymes in energy conservation; however, their effect on cell behavior is poorly understood. Pseudomonas aeruginosa can perform both aerobic respiration and denitrification. Previous studies demonstrated that cbb₃-type cytochrome c oxidases that support aerobic respiration are more highly expressed in P. aeruginosa under anoxic conditions than are other aerobic respiratory enzymes. However, little is known about their role under such conditions. In this study, it was shown that cbb₃ oxidases of P. aeruginosa PAO1 alter anaerobic growth, the denitrification process, and cell morphology under anoxic conditions. Furthermore, biofilm formation was promoted by the cbb₃ oxidases under anoxic conditions. cbb₃ oxidases led to the accumulation of nitric oxide (NO), which is produced during denitrification. Cell elongation induced by NO accumulation was reported to be required for robust biofilm formation of P. aeruginosa PAO1 under anoxic conditions. Our data show that cbb₃ oxidases promote cell elongation by inducing NO accumulation during the denitrification process, which further leads to robust biofilms. Our findings show that cbb₃ oxidases, which have been well studied as aerobic respiratory enzymes, are also involved in denitrification and influence the lifestyle of P. aeruginosa PAO1 under anoxic conditions.     

ESR study on synthetic glyceroglycolipid liposomal membranes

We previously reported that glyceroglycolipid liposomes without cholesterol activated mouse peritoneal macrophages in vivo and in vitro, whereas glyceroglycolipid liposomes containing equimolar cholesterol did not. In order to characterize the properties of the glyceroglycolipid membranes, ESR spectroscopic studies were carried out with an acyl spin-labeled galactosyl ceramide (SL-GC) or a headgroup spin-labeled phospholipid (SL-6-DPPA) in 1,2-dipalmitoyl[beta-cellobiosyl-(1'---3)]glycerol (Cel-DAG) liposomal membranes. The ESR spectrum of the SL-GC in the Cel-DAG liposomes at 37 degrees C was a single broad line, indicating that the SL-GC molecules were excluded almost completely from Cel-DAG domains and formed clusters in the membranes. The spectrum of SL-6-DPPA in the Cel-DAG liposomes at 37 degrees C showed broad resonance lines with the central peak being the highest, while that at 60 degrees gave narrow lines with the low-field peak being the highest. This observation and rotational correlation time analysis showed that the molecular motions of spin-label moiety of the SL-6-DPPA were extremely restricted at 37 degrees C but not above Tc. These results suggest that below Tc the Cel-DAG molecules are packed tightly and restricted in motion in the membrane. Incorporation of cholesterol into the Cel-DAG liposomal membranes gave (1) the spectra of the SL-GC triplet, and (2) the spectra of the SL-6-DPPA narrow resonance with the low-field peak being the highest. These results suggest that cholesterol disturbs the rigid-packed structure of the Cel-DAG membrane and increases the molecular motions of the Cel-DAG. The DSC analysis of Cel-DAG with and without cholesterol agreed well to the results of the ESR technique. Thus we assume that peritoneal macrophages recognize the rigid-packed carbohydrate residues which are restricted in motion on the Cel-DAG membranes.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.