ß-ureidopropionase deficiency: phenotype, genotype and protein structural consequences in 16 patients

?-ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-?-alanine and N-carbamyl-?-aminoisobutyric acid to ?-alanine and ?-aminoisobutyric acid, ammonia and CO(2). To date, only five genetically confirmed patients with a complete ?-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified ?-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-?-alanine and N-carbamyl-?-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding ?-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant ?-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ?-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ?-ureidopropionase patients, indicating that ?-ureidopropionase deficiency may be more common than anticipated.     

Salt tolerance of a cash crop halophyte Suaeda fruticosa: biochemical responses to salt and exogenous chemical treatments

Suaeda fruticosa Forssk is a leaf succulent obligate halophyte that produces numerous seeds under saline conditions. Seeds are a good source of high quality edible oil and leaves are capable of removing substantial amount of salt from the saline soil besides many other economic usages. Little is known about the biochemical basis of salt tolerance in this species. We studied some biochemical responses of S. fruticosa to different exogenous treatments under non-saline (0 mM), moderate (300 mM) or high (600 mM) NaCl levels. Eight-week-old seedlings were sprayed twice a week with distilled water, hydrogen peroxide (H₂O₂, 100 μM), glycine betaine (GB, 10 mM), or ascorbic acid (AsA, 20 mM) for 30 days. At moderate (300 mM) NaCl, leaf Na⁺, Ca²⁺ and osmolality increased, along with unchanged ROS and antioxidant enzyme activities, possibly causing a better plant growth. Plants grew slowly at 600 mM NaCl to avoid leaf Na⁺ buildup relative to those at 300 mM NaCl. Exogenous application of distilled water and H₂O₂ improved ROS scavenging mechanisms, although growth was unaffected. ASA and GB alleviated salt-induced growth inhibition at 600 mM NaCl through enhancing the antioxidant defense system and osmotic and ion homeostasis, respectively.     

An immunohistochemical method for identifying fibroblasts in formalin-fixed, paraffin-embedded tissue.

Fibroblasts are critical for tissue homeostasis, and their inappropriate proliferation and activation can result in common and debilitating conditions including fibrosis and cancer. We currently have a poor understanding of the mechanisms that control the growth and activation of fibroblasts in vivo, in part because of a lack of suitable fibroblast markers. We have taken advantage of an antibody previously shown to stain stromal cells in frozen tissues (TE-7) and identified conditions in which it can be used to stain fibroblasts and myofibroblasts in the paraffin-embedded tissue samples routinely collected for pathological analysis. We show that this antibody recognizes growing and quiescent fibroblasts and myofibroblasts by immunohistochemistry, immunofluorescence, and ELISA assays. We also present its staining patterns in normal tissue samples and in breast tumors.     

Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders

Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 mul) were hydrolyzed, extracted, and derivatized with DAABD-AE in approximately 2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.     

Field site selection: getting it right first time around

The selection of suitable field sites for integrated control of Anopheles mosquitoes using the sterile insect technique (SIT) requires consideration of the full gamut of factors facing most proposed control strategies, but four criteria identify an ideal site: 1) a single malaria vector, 2) an unstructured, relatively low density target population, 3) isolation of the target population and 4) actual or potential malaria incidence. Such a site can exist in a diverse range of situations or can be created. Two contrasting SIT field sites are examined here: the desert-flanked Dongola Reach of the Nile River in Northern State, Sudan, where malaria is endemic, and the island of La Reunion, where autochthonous malaria is rare but risk is persistent. The single malaria-transmitting vector at both sites is Anopheles arabiensis. In Sudan, the target area is a narrow 500 km corridor stretching from the rocky terrain at the Fourth Cataract - just above the new Merowe Dam, to the northernmost edge of the species range, close to Egypt. Vector distribution and temporal changes in density depend on the Nile level, ambient temperature and human activities. On La Reunion, the An. arabiensis population is coastal, limited and divided into three areas by altitude and exposure to the trade winds on the east coast. Mosquito vectors for other diseases are an issue at both sites, but of primary importance on La Reunion due to the recent chikungunya epidemic. The similarities and differences between these two sites in terms of suitability are discussed in the context of area-wide integrated vector management incorporating the SIT.     

Ethical,legal and social aspects of the approach in Sudan

The global malaria situation, especially in Africa, and the problems frequently encountered in chemical control of vectors such as insecticide resistance, emphasize the urgency of research, development and implementation of new vector control technologies that are applicable at regional and local levels. The successful application of the sterile insect technique (SIT) for the control of the New World screwworm Cochliomyia hominivorax and several species of fruit flies has given impetus to the use of this method for suppression or elimination of malaria vectors in some areas of Africa including Northern State of Sudan. The research and development phase of the Northern State feasibility study has been started. Sudanese stakeholders are working side-by-side with the International Atomic Energy Agency in the activities of this important phase. Several ethical, legal and social issues associated with this approach arose during this phase of the project. They need to be seriously considered and handled with care. In this paper, these issues are described, and the current and proposed activities to overcome potential hurdles to ensure success of the project are listed.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

Ethnopharmacological studies of indigenous plants in Kel village,Neelum Valley,Azad Kashmir,Pakistan

Background

This explorative study was undertaken for the first time in Kel village located in the Upper Neelum Valley, Azad Kashmir, Pakistan. The purpose was to document the indigenous knowledge of the native people used in the preparation of herbal medicines.

Methods

To get the data on traditional uses of medicinal plants, 20 informants were interviewed. Quantitative ethnobotanical indices, i.e., use value (UV), relative frequencies of citation (RFC), informant consensus factor (Fic), fidelity level (FL), data matrix ranking (DMR), preference ranking (PR), and jaccard index (JI), were calculated for the recorded medicinal plants.

Results

A total of 50 medicinal plants belonging to 33 families used in 13 disease categories were documented. Leaves were the frequently used plant parts, and decoction was the commonly used method for herbal medicine. Plants with high use value were Berberis lycium (2.05), Impatiens glandulifera (1.95), Artemisia scoparia (1.75), Ageratum conozoides (1.75), and Achillea millefolium (1.7). The highest RFC value was calculated for Berberis lycium (0.75), Cynoglossum lanceolatum (0.65), and Impatiens glandulifera and Achillea millefolium (0.60 each). The maximum informant consensus factor was for urinary system, cardiac diseases, baldness, and abortion and miscarriage (1.00). Berberis lyceum (95%) used in jaundice, hepatitis, typhoid, fever, and tuberculosis disorders. Plants with maximum fidelity level (FL) were Berberis lycium (95%) followed by Dioscorea bulbifera, Impatiens glandulifera, and Artemisia vulgaris (90%). Olea ferruginea was the most multipurpose plant and exports (21.2%) was the leading threat in the area. The pearson correlation coefficient (0.500) showed a positive correlation between the use value and relative frequency of citation.

Conclusion

The present study provides useful information about traditional uses of medicinal plants used by local communities in different ailments. The plants with the highest use values could be employed in pharmacological research and biotechnological approaches in order to achieve adequate revenue. Some of the plants in the study area are facing high threats of becoming rare, and conservation initiatives are needed to conserve them for sustainable management in the region.