Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Molecular cloning of chicken prepro-orexin cDNA and preferential expression in the chicken hypothalamus

Chicken prepro-orexin cDNA has been cloned, sequenced and characterized. The predicted amino acid sequence of chicken prepro-orexin cDNA revealed that orexin-A and -B are highly conserved among vertebrate species. In situ hybridization and immunohistochemistry localized orexin-positive cell bodies in the periventricular hypothalamic nucleus extending into the lateral hypothalamic area. Comparisons of orexin gene expression in the brains of 24-h-fasted and ad libitum-fed chickens were made using semi-quantitative RT-PCR. No significant differences in orexin mRNA expression were observed.     

An application of bacterial flow cytometry: Evaluation of the toxic effects of four heavy metals on Acinetobacter sp. with potential for bioremediation of contaminated wastewaters

Acinetobacter johnsonii has potential use in the remediation of heavy metal-contaminated wastewaters. For metal accumulation, cells must remain intact and metabolically active. The effect of possible accumulation targets, Cd²⁺, UO₂ ²⁺, Cu²⁺, and Ni²⁺ on cytoplasmic membrane integrity and polarity was investigated by flow cytometry using a mixture of two fluorescent dyes, propidium iodide and bis-oxonol. The former binds to DNA but cannot cross an intact cytoplasmic membrane, whilst the latter is anionic, lipophilic and stains depolarized cytoplasmic membranes. All four metals permeabilized the cytoplasmic membrane of some cells during the period of exposure. The effects of the metals differed in that Cu²⁺ and Cd²⁺ also generated an intermediate population of cells, having intact but depolarized cytoplasmic membranes. Electron microscopy showed corresponding cellular abnormalities following metal exposure. © Rapid Science Ltd. 1998     

Derivative emission spectrofluorimetry: Application to the analysis of newly approved FDA combination of ibuprofen and famotidine in tablets

A new combination of ibuprofen (NSAID) and famotidine (H₂ receptor antagonist) was recently approved by the FDA. It was formulated to relief pain while decreasing the risk of ulceration, which is a common problem for patients receiving NSAID. A rapid and simple derivative emission spectrofluorimetric method is proposed for the simultaneous analysis of this combination in their pharmaceutical preparation. The method is based upon measurement of the native fluorescence intensity of the two drugs at λ_ex = 233 nm in acetonitrile. The emission data were differentiated using the first (D1) derivative technique. The plots of derivative fluorescence intensity versus concentration were rectilinear over a range of 2–35 and 0.4–8 µg/mL for both ibuprofen (IBU) and famotidine (FAM), respectively. The method was sensitive as the limits of detection were 0.51 and 0.12 µg/mL and limits of quantitation were 1.70 and 0.39 µg/mL, for IBU and FAM respectively. The proposed derivative emission spectrofluorimetric method was successfully applied for the determination of the two drugs in their synthetic mixtures and tablets with good accuracy and precision. The proposed method was validated as per ICH guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

Mycetoma in the Sudan: An Update from the Mycetoma Research Centre,University of Khartoum,Sudan

This communication reports on the Mycetoma Research Centre of the University of Khartoum, Sudan experience on 6,792 patients seen during the period 1991–2014.The patients were predominately young (64% under 30 years old) males (76%). The majority (68%) were from the Sudan mycetoma belt and 28% were students. Madurella mycetomatis eumycetoma was the most common type (70%). In 66% of the patients the duration of the disease was less than five years, and 81% gave a history of sinuses discharging mostly black grains (78%). History of trauma at the mycetoma site was reported in 20%. Local pain was reported in 27% of the patients, and only 12% had a family history of mycetoma. The study showed that 57% of the patients had previous surgical excisions and recurrence, and only 4% received previous medical treatment for mycetoma. Other concomitant medical diseases were reported in 4% of the patients. The foot (76%) and hand (8%) were the most commonly affected sites. Less frequently affected sites were the leg and knee (7%), thigh (2%), buttock (2%) and arm and forearm (1%). Rare sites included the chest wall, head and neck, back, abdominal wall, perineum, oral cavity, tongue and eye. Multiple sites mycetoma was recorded in 135 (2%) of cases. At presentation, 37% of patients had massive lesions, 79% had sinuses, 8% had local hyper-hydrosis at the mycetoma lesion, 11% had regional lymphadenopathy, while 6% had dilated tortuous veins proximal to the mycetoma lesions. The diagnosis of mycetoma was established by combined imaging techniques and cytological, histopathological, serological tests and grain culture. Patients with actinomycetoma received a combination of antimicrobial agents, while eumycetoma patients received antifungal agents combined with various surgical excisions. Surgical excisions in the form of wide local excision, debridement or amputation were done in 807 patients, and of them 248 patients (30.7%) had postoperative recurrence. Different types of amputations were done in 120 patients (1.7%).     

Head and Neck Mycetoma: The Mycetoma Research Centre Experience

Mycetoma is a unique neglected tropical disease which is endemic in what is known as the “mycetoma belt”. The disease has many devastating impacts on patients and communities in endemic area and is characterised by massive deformity, destruction and disability. Mycetoma is commonly seen in the foot and hand and less frequent in other parts of the body. Mycetoma of the head and neck is a rarity and is associated with high morbidity and even mortality if not treated early. In this communication we report on 49 patients with head and neck mycetoma followed up at the Mycetoma Research Centre in Khartoum. Most of the reported patients had actinomycetoma and the majority were young adult males from mycetoma endemic areas in the Sudan. Most of them were students, farmers and workers. Prior to presentation the majority had long disease duration and the cause was multifactorial. Advanced disease with massive lesion, deformity and disability was the common presentation. There was no obvious history of local trauma, familial tendency or other predisposing factor identified in this group of patients. MRI and CT scan were the most accurate diagnostic tools to determine the disease extent. The treatment outcome was rather poor and characterised by low cure rate, poor outcome and high follows-up dropout. Such a gloomy outcome calls for structured and objective health education programs.     

Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay

Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay.     

A new genus of African Acrometopini (Tettigoniidae: Phaneropterinae) based on morphology,chromosomes, acoustics,distribution, and molecular data,and the description of a new species

A new genus, Altihoratosphaga, is erected for species formerly assigned to Horatosphaga Schaum, 1853, and a new species is described. Four species are included in Altihoratosphaga: Altihoratosphaga nomima (Karsch, 1896), Altihoratosphaga montivaga ( Sjöstedt, 1909 ), Altihoratosphaga nou (Hemp, 2007) and Altihoratosphaga hanangensis sp. nov. All four species are restricted to Tanzanian localities, and, except for A. nomima, for which no ecological data are available, are confined to montane forest habitats. Data on ecology, acoustics, chromosomes, and molecular relationships are provided, as well as a key to Altihoratosphaga species. The present‐day distribution of Altihoratosphaga species suggests former migration events at times when wetter and colder climatic fluctuations favoured connections between montane forest communities, which today are isolated, enabling flightless taxa such as Altihoratosphaga and Monticolaria to spread. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 66–82.