Elucidation of ATP-stimulated stress protein expression of RBA-2 type-2 astrocytes: ATP potentiate HSP60 and Cu/Zn SOD expression and stimulates pI shift of peroxiredoxin II

ATP has been shown to mediate stress responses in the brain. The present study examined the ATP-stimulated stress protein expression of RBA-2 type-2 astrocytes. Our results revealed that ATP stimulated HSP60 expression in a dose- and time-dependent manner. The stimulation requires a minimal ATP concentration of 500 microM and high concentration of extracellular ATP (1 mM) stimulated a significant increase of HSP60 expression from 2 to 24 h. In addition, the ATP-stimulated HSP60 expressions were inhibited by inhibitors for protein kinase C (PKC) and phospholipase D (PLD), and by antioxidants, resveratrol, and catalase. Furthermore, ATP stimulated the expression of Cu/Zn superoxide dismutase (SOD). In addition, ATP and P2X7 receptor selective agonist BzATP also decreased mitochondria membrane potential measured by flow cytometry. To further examine the proteins involving in ATP-mediated stress responses, we conducted proteomic analysis. We found that RBA-2 astrocytes possess abundant peroxiredoxin II (Prx II), an antioxidant enzyme. ATP and exogenous H2O2 stimulated Prx II shifting from oxidized form to reduced form. Thus, we concluded that ATP potentiated the expression of HSP60 and Cu/Zn SOD, and decreased mitochondria membrane potential. In addition, RBA-2 astrocytes expressed Prx II that might also serve as a protective mechanism to control the concentration of reactive oxygen species.     

Unexpected NRY chromosome variation in Northern Island Melanesia

To investigate the paternal population history of populations in Northern Island Melanesia, 685 paternally unrelated males from 36 populations in this region and New Guinea were analyzed at 14 regionally informative binary markers and 7 short tandem repeat (STR) loci from the nonrecombining portion of the Y chromosome. Three newly defined binary markers (K6-P79, K7-P117, and M2-P87) aided in identifying considerable heterozygosity that would have otherwise gone undetected. Judging from their geographic distributions and network analyses of their associated STR profiles, 4 lineages appear to have developed in this region and to be of considerable age: K6-P79, K7-P117, M2-P87, and M2a-P22. The origins of K5-M230 and M-M4 are also confirmed as being located further west, probably in New Guinea. In the 25 adequately sampled populations, the number of different haplogroups ranged from 2 in the single most isolated group (the Aita of Bougainville), to 9, and measures of molecular diversity were generally not particularly low. The resulting pattern contradicts earlier findings that suggested far lower male-mediated diversity and gene exchange rates in the region. However, these earlier studies had not included the newly defined haplogroups. We could only identify a very weak signal of recent male Southeast Asian genetic influence (<10%), which was almost entirely restricted to Austronesian (Oceanic)-speaking groups. This contradicts earlier assumptions on the ancestral composition of these groups and requires a revision of hypotheses concerning the settlement of the islands of the central Pacific, which commenced from this region.     

Laccase-induced derivatization of unprotected amino acid L-tryptophan by coupling with p-hydroquinone 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide

Summary. We have studied the enzymatic derivatization of amino acids by use of the polyphenol oxidase laccase. Derivatization of L-tryptophan was achieved by enzymatic crosslinking with the laccase substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. The main product (yield up to 70%) was identified as the quinoid compound 2-[2-(2-hydroxy-ethylcarbamoyl)-3,6-dioxo-cyclohexa-1,4-dienylamino]-3-(1H-indol-3-yl)- propionic acid and demonstrates that laccase-catalyzed C–N-coupling occurred on the amino group of the aliphatic side chain. These enzyme based reactions provide a simple and fast method for the derivatization of unprotected amino acids.     

Cancer mortality among German aircrew: second follow-up

Aircrew members are exposed to cosmic radiation and other specific occupational factors. In a previous analysis of a large cohort of German aircrew, no increase in cancer mortality or dose-related effects was observed. In the present study, the follow-up of this cohort of 6,017 cockpit and 20,757 cabin crew members was extended by 6 years to 2003. Among male cockpit crew, the resulting all-cancer standardized mortality ratio (SMR) (n = 127) is 0.6 (95% CI 0.5–0.8), while for brain tumors it is 2.1 (95% CI 1.0–3.9). The cancer risk is significantly raised (RR = 2.2, 95% CI 1.2–4.1) among cockpit crew members employed 30 years or more compared to those employed less than 10 years. Among both female and male cabin crew, the all-cancer SMR and that for most individual cancers are close to 1. The SMR for breast cancer among female crew is 1.2 (95% CI 0.8–1.8). Non-Hodgkin’s Lymphoma among male cabin crew is increased (SMR 4.2; 95% CI 1.3–10.8). However, cancers associated with radiation exposure are not raised in the cohort. It is concluded that among cockpit crew cancer mortality is low, particularly for lung cancer. The positive trend of all cancer with duration of employment persists. The increased brain cancer SMR among cockpit crew requires replication in other cohorts. For cabin crew, cancer mortality is generally close to population rates. Cosmic radiation dose estimates will allow more detailed assessments, as will a pooling of updated aircrew studies currently in planning.     

Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques

Bacillus subtilis has been developed as a model system for physiological proteomics. However, thus far these studies have mainly been limited to cytoplasmic, extracellular, and cell-wall attached proteins. Although being certainly important for cell physiology, the membrane protein fraction has not been studied in comparable depth due to inaccessibility by traditional 2-DE-based workflows and limitations in reliable quantification. In this study, we now compare the potential of stable isotope labeling with amino acids (SILAC) and (14)N/(15)N-labeling for the analysis of bacterial membrane fractions in physiology-driven proteomic studies. Using adaptation of B. subtilis to amino acid (lysine) and glucose starvation as proof of principle scenarios, we show that both approaches provide similarly valuable data for the quantification of bacterial membrane proteins. Even if labeling with stable amino acids allows a more straightforward analysis of data, the (14)N/(15)N-labeling has some advantages in general such as labeling of all amino acids and thereby increasing the number of peptides for quantification. Both, SILAC as well as (14)N/(15)N-labeling are compatible with 2-DE, 2-D LC-MS/MS, and GeLC-MS/MS and thus will allow comprehensive simultaneous interrogation of cytoplasmic and enriched membrane proteomes.     

Adaptation of sorghum: characterisation of genotypic flowering responses to temperature and photoperiod

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop grown in a wide range of tropical and temperate environments. This study was conducted to characterise the photothermal flowering responses of sorghum genotypes and to examine relationships between photothermal characteristics and environment of origin in order to better understand the phenological basis of adaptation to environment in sorghum. Twenty-four germplasm accessions and one hybrid from 24 major sorghum-growing areas were grown in a wide range of environments varying in temperature and photoperiod in India, Kenya and Mali between 1992 and 1995. Times from sowing to flowering (f) were recorded, and the responsiveness of 1/f to temperature and photoperiod was quantified using photothermal models. Times from sowing to flowering were accurately predicted in a wide range of environments using a multiplicative rate photothermal model. Significant variation in the minimum time to flower (F_m) and photoperiod sensitivity (critical photoperiod, P_c, and photoperiod-sensitivity slope, P_s) was observed among the genotypes; in contrast there was little variation in base temperature (T_b). Adaptation of sorghum to the diverse environments in which it is grown was largely determined by photoperiod sensitivity and minimum time to flower; photoperiod sensitivity determines broad adaptation to latitude (daylength), while variation in the minimum time to flower determines specific adaptation within smaller ranges of latitude, e.g. within the humid and sub-humid tropics. Received: 16 January 1999 / Accepted: 11 March 1999     

Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.

Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. Here, we report the requirement for two other proteins, cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1.     

Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean and ob/ob mice

Liver X receptor (LXR) agonists have been proposed to act as anti-diabetic drugs. However, pharmacological LXR activation leads to severe hepatic steatosis, a condition usually associated with insulin resistance and type 2 diabetes mellitus. To address this apparent contradiction, lean and ob/ob mice were treated with the LXR agonist GW-3965 for 10 days. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Hepatic glucose production (HGP) and metabolic clearance rate (MCR) of glucose were determined with stable isotope techniques. Blood glucose and hepatic and whole body insulin sensitivity remained unaffected upon treatment in lean mice, despite increased hepatic triglyceride contents (61.7 +/- 7.2 vs. 12.1 +/- 2.0 nmol/mg liver, P < 0.05). In ob/ob mice, LXR activation resulted in lower blood glucose levels and significantly improved whole body insulin sensitivity. GW-3965 treatment did not affect HGP under normo- and hyperinsulinemic conditions, despite increased hepatic triglyceride contents (221 +/- 13 vs. 176 +/- 19 nmol/mg liver, P < 0.05). Clamped MCR increased upon GW-3965 treatment (18.2 +/- 1.0 vs. 14.3 +/- 1.4 ml x kg(-1) x min(-1), P = 0.05). LXR activation increased white adipose tissue mRNA levels of Glut4, Acc1 and Fasin ob/ob mice only. In conclusion, LXR-induced blood glucose lowering in ob/ob mice was attributable to increased peripheral glucose uptake and metabolism, physiologically reflected in a slightly improved insulin sensitivity. Remarkably, steatosis associated with LXR activation did not affect hepatic insulin sensitivity.