Live-cell assays to identify regulators of ER-to-Golgi trafficking

We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (β1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.     

Land Use and Land Right Dynamics—Determinants for Resource Management Options in Eastern Burkina Faso

The paper first examines established theories on the population-environmental nexus. Farmers' options in responding to declining soil fertility or shortage of land vary from village to village and even household to household. Responses are conditioned by social relations with neighboring villages and the labor availability of individual households. The example from the Boulgou Province in Burkina Faso shows that some farmers have been able to expand their acreage in other villages' territories on a virtually permanent basis, thereby compensating for increased population pressure and for land degradation. Currently, labor availability, social relations, and distance to the land seem to be the main constraints on land expansion. However, the pattern of land use changes is expected to be increasingly influenced by the existence of more formally established rights than at present. In the light of the empirical findings, the relevance of the Gestion des Terroir Villagois concept as a tool for planning for sustainable natural resource management at the village level is discussed. This approach seems imperfectly suited to address a reality in which social and physical environments are superimposed in a spatially complex way.     

A variety of volatile compounds as markers in unifloral honey from dalmatian sage (Salvia officinalis L.)

Volatile compounds of unifloral Salvia officinalis L. honey has been investigated for the first time. The botanical origin of ten unifloral Salvia honey samples has been ascertained by pollen analysis (the honey samples displayed 23-60% of Salvia pollen). Fifty-four volatile compounds were identified by GC and GC/MS in ten Salvia honey extracts obtained by ultrasound-assisted extraction (USE) with pentane/Et(2)O 1 : 2. The yield of isolated volatiles varied from 25.7 to 30.5 mg kg(-1). Salvia honey could be distinguished on the basis of the high percentage of benzoic acid (6.4-14.8%), and especially phenylacetic acid (5.7-18.4%). Minor, but floral-origin important volatiles were identified such as shikimate pathway derivatives, 'degraded-carotenoid-like' structures (3,5,5-trimethylcyclohex-2-ene derivatives) and 2,6,6-trimethylcyclohex-2-ene derivatives. Compounds from other metabolic pathways such as aliphatic acids and higher linear hydrocarbons, as well as heterocycles (pyrans, furans, and pyrroles), were also present. Most of the identified compounds do not constitute specific Salvia honey markers, due to their presence in honeys of other botanical origins; however, their ratio in different honeys could be useful to distinguish floral origin. Salvia-honey volatile markers were: benzoic acid, phenylacetic acid, p-anisaldehyde, alpha-isophorone, 4-ketoisophorone, dehydrovomifoliol, 2,6,6-trimethyl-4-oxocyclohex-2-ene-1-carbaldehyde, 2,2,6-trimethylcyclohexane-1,4-dione, and coumaran.     

The Volatile Profiles of a Rare Apple (Malus domestica Borkh.) Honey: Shikimic Acid‐Pathway Derivatives,Terpenes, and Others

The volatile profiles of rare Malus domestica Borkh . honey were investigated for the first time. Two representative samples from Poland (sample I) and Spain (sample II) were selected by pollen analysis (44–45% of Malus spp. pollen) and investigated by GC/FID/MS after headspace solid‐phase microextraction (HS‐SPME) and ultrasonic solvent extraction (USE). The apple honey is characterized by high percentage of shikimic acid‐pathway derivatives, as well as terpenes, norisoprenoids, and some other compounds such as coumaran and methyl 1H‐indole‐3‐acetate. The main compounds of the honey headspace were (sample I; sample II): benzaldehyde (9.4%; 32.1%), benzyl alcohol (0.3%; 14.4%), hotrienol (26.0%, 6.2%), and lilac aldehyde isomers (26.3%; 1.7%), but only Spanish sample contained car‐2‐en‐4‐one (10.2%). CH₂Cl₂ and pentane/Et₂O 1 : 2 (v/v) were used for USE. The most relevant compounds identified in the extracts were: benzaldehyde (0.9–3.9%), benzoic acid (2.0–11.2%), terpendiol I (0.3–7.4%), coumaran (0.0–2.8%), 2‐phenylacetic acid (2.0–26.4%), methyl syringate (3.9–13.1%), vomifoliol (5.0–31.8%), and methyl 1H‐indole‐3‐acetate (1.9–10.2%). Apple honey contained also benzyl alcohol, 2‐phenylethanol, (E)‐cinnamaldehyde, (E)‐cinnamyl alcohol, eugenol, vanillin, and linalool that have been found previously in apple flowers, thus disclosing similarity of both volatile profiles.     

Antioxidant Capacity and Chemical Profiles of Satureja montana L. Honey: Hotrienol and Syringyl Derivatives as Biomarkers

The present study is focused on the antioxidant capacity and chemical profiling of eight Croatian Satureja montana L. honey samples. Among the 20 compounds obtained by headspace solid‐phase microextraction (HS‐SPME) and identified by GC‐FID and GC/MS analyses, hotrienol was predominant (75.9–81.7%). The honey matrix volatile/semivolatile profile was investigated by ultrasonic solvent extraction (USE) followed by GC‐FID and GC/MS analyses. The major compounds identified by this latter method were the sinapic‐acid derivatives methyl syringate (36.2–72.8%) and syringaldehyde (2.2–43.1%). Direct, targeted HPLC‐DAD analyses of the native honey samples revealed the presence of methyl syringate (7.10–39.60 mg/kg) and syringic acid (0.10–1.70 mg/kg). In addition, the total phenolic content of the samples was determined by the Folin? Ciocalteu assay (311.0–465.9 mg GAE/kg), and the antioxidant capacity was evaluated by the DPPH radical‐scavenging activity (0.5–1.0 mmol TEAC/kg) and the ferric reducing antioxidant power (2.5–5.1 mmol Fe²⁺/kg).     

Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like modifier activating enzyme 6, Uba6

Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.     

International NGOs and Land Use Change in a Southern Highland Region of Ecuador

Nongovernmental organizations are increasingly influencing resource management and land use in areas of small farm agriculture in Latin America. A field study of NGOs working in upper Canar, a region in the southern highlands of Ecuador, documents the changing human environment relations in an indigenous area and the influence of NGOs in the change process. Case studies of PLAN International and CARE indicate that the NGOs are helping marginalized producers shift land use away from traditional grains and tubers toward dairying and vegetables. Given current needs and resource constraints, the new land uses represent effective adaptive strategies. However, the NGO work is having notable consequences for land use intensity and labor utilization patterns.