Enzymatic radioiodination of phospholipids catalyzed by lactoperoxidase

Phospholipids were iodinated with iodide by a lactoperoxidase-catalyzed reaction in the presence of controlled amounts of H₂O₂ which were continuously supplied by glucose oxidase + glucose. Different molecular and ionic species of inorganic iodine present in the reaction mixture (i.e., I^?, I₂, I₃^?) were eliminated by thiosulfate reduction to I^? followed by gel filtration on Sephadex LH-20 which separated I^? from the phospholipids completely. Final separation and identification of individual phospholipids were done on a column of silica gel H using a single solvent mixture consisting of CHCl₃:CH₃OH:CH₃COOH:H₂O (25:15:4:2, by volume). Application of phospholipases A₂ and D or transesterification provided evidence to indicate a covalent iodination of the fatty acid moiety of the lipids by the enzymatic process, which apparently is substitution but could also proceed by addition to the double bonds, when present.     

Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria

Primaquine and tafenoquine are the only licensed drugs with activity against Plasmodium vivax hypnozoites but cause haemolysis in patients with glucose–6–phosphate dehydrogenase (G6PD) deficiency. Malaria also causes haemolysis, leading to the replacement of older erythrocytes with low G6PD activity by reticulocytes and young erythrocytes with higher activity. Aim of this study was to assess the impact of acute malaria on G6PD activity. Selected patients with uncomplicated malaria were recruited in Bangladesh (n = 87), Indonesia (n = 75), and Ethiopia (n = 173); G6PD activity was measured at the initial presentation with malaria and a median of 176 days later (range 140 to 998) in the absence of malaria. Among selected participants (deficient participants preferentially enrolled in Bangladesh but not at other sites) G6PD activity fell between malaria and follow up by 79.1% (95%CI: 40.4 to 117.8) in 6 participants classified as deficient (<30% activity), 43.7% (95%CI: 34.2 to 53.1) in 39 individuals with intermediate activity (30% to <70%), and by 4.5% (95%CI: 1.4 to 7.6) in 290 G6PD normal (≥70%) participants. In Bangladesh and Indonesia G6PD activity was significantly higher during acute malaria than when the same individuals were retested during follow up (40.9% (95%CI: 33.4–48.1) and 7.4% (95%CI: 0.2 to 14.6) respectively), whereas in Ethiopia G6PD activity was 3.6% (95%CI: -1.0 to -6.1) lower during acute malaria. The change in G6PD activity was apparent in patients presenting with either P. vivax or P. falciparum infection. Overall, 66.7% (4/6) severely deficient participants and 87.2% (34/39) with intermediate deficiency had normal activities when presenting with malaria.These findings suggest that G6PD activity rises significantly and at clinically relevant levels during acute malaria. Prospective case-control studies are warranted to confirm the degree to which the predicted population attributable risks of drug induced haemolysis is lower than would be predicted from cross sectional surveys.     

Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.     

Cellular effects of electromagnetic fields

Studies at the cellular level are needed to reveal the cellular and molecular biological mechanisms underlying the biological effects and possible health implications of non-ionising radiation, such as extremely low frequency (ELF) magnetic fields (MFs) and radiofrequency (RF) fields. Our research group has studied the effects of 50 Hz ELF MFs (caused by power lines and electric devices) and 872 MHz or 900 MHz RFs (emitted by mobile phones and their base stations) on cellular ornithine decarboxylase activity, cell cycle kinetics, cell proliferation, and necrotic or apoptotic cell death. For RFs, pulse-modulated (217 Hz modulation frequency corresponding a global system for mobile communication-type signal) or continuous wave (unmodulated) signals were used. To expose the cell cultures to MFs or RFs, specially developed exposure systems were used, where levels of electromagnetic field exposure and the conditions of cell culture could be precisely controlled. A coexposure approach was used in many studies, i.e. the cell cultures were exposed to other stressors in addition to MFs or RFs. Ultraviolet radiation, serum deprivation, or fresh medium addition, were used as co-exposures. The results presented in this short review show that the effects of mere MFs or RF on cell culture models are quite minor, but that various co-exposure approaches warrant additional study.     

Progesterone and the metabolic control of the lactose biosynthetic pathway during lactogenesis in the rat

1. Lactogenesis was initiated in pregnant rats by ovariectomy, thereby causing progesterone withdrawal, after which the mammary tissue was analysed for contents of enzymes and metabolites concerned with the biosynthesis of lactose. 2. Lactose synthesis increased about 126-fold with little or no accompanying change in the contents of most metabolic intermediates or in the adenine nucleotide energy charge. 3. Comparison of mass-action ratios with equilibrium constants showed that phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDP-glucose epimerase (EC 5.1.3.2.) catalysed reactions close to equilibrium. Nucleoside diphosphokinase (EC 2.7.4.6.) activity was very high and probably equilibrates the UTP-UDP and ATP-ADP couples. Lactose synthetase and hexokinase (EC 2.7.1.1) appeared to catalyse rate-limiting reactions. 4. Large increases were seen of UDP-glucose pyrophosphorylase (5-fold), lactose synthetase A protein (3.8-fold) and alpha-lactalbumin (28-fold), but not of hexokinase, phosphoglucomutase, UDP-glucose epimerase, nucleoside diphosphokinase or glucose 6-phosphate dehydrogenase (EC 1.1.1.49) activities. 5. It appeared that the increased lactose synthesis was largely accounted for by the increased lactose synthetase A protein activity and alpha-lactalbumin.     

Thermodynamics of Cd2+ and Zn2+ binding by the phytochelatin (gamma-Glu-Cys)4-Gly and its precursor glutathione

Isothermal titration calorimetry (ITC) was used to study the binding of Cd(2+) and Zn(2+) by glutathione (GSH) and phytochelatins (PC(n)), the metal sequestering compounds in plants and algae. The results are compared with those obtained by differential pulse polarography (DPP) assisted by multivariate curve resolution with alternating least squares (MCR-ALS) and by electrospray ionization mass spectrometry (ESI-MS). ITC allows one to determine (i) the stoichiometries of the different complexes (and confirms those found by DPP/MCR-ALS and ESI-MS) and (ii) their binding and thermodynamic parameters. For Cd-PC(4), the sequential binding sites model with two identical sites yields the best fitting of ITC curves and confirms the presence of CdPC(4) and Cd(2)PC(4) complexes. For Zn-PC(4), exothermic formation of ZnPC(4) is reported. Conditional stability and formation constants for Cd-GSH and Zn-GSH are determined from the fitting of the proper model to experimental ITC curves. The effect of different buffers in the complexation processes shows the key role of the choice of the buffer in calorimetric study.     

The role of heterogenous environmental conditions in shaping the spatiotemporal distribution of competing Aedes mosquitoes in Panama: implications for the landscape of arboviral disease transmission

Monitoring the invasion process of the Asian tiger mosquito Aedes albopictus and its interaction with the contender Aedes aegypti, is critical to prevent and control the arthropod-borne viruses (i.e., Arboviruses) they transmit to humans. Generally, the superior ecological competitor Ae. albopictus displaces Ae. aegypti from most geographic areas, with the combining factors of biology and environment influencing the competitive outcome. Nonetheless, detailed studies asserting displacement come largely from sub-tropical areas, with relatively less effort being made in tropical environments, including no comprehensive research about Aedes biological interactions in Mesoamerica. Here, we examine contemporary and historical mosquito surveillance data to assess the role of shifting abiotic conditions in shaping the spatiotemporal distribution of competing Aedes species in the Republic of Panama. In accordance with prior studies, we show that Ae. albopictus has displaced Ae. aegypti under suboptimal wet tropical climate conditions and more vegetated environments within the southwestern Azuero Peninsula. Conversely, in the eastern Azuero Peninsula, Ae. aegypti persists with Ae. albopictus under optimal niche conditions in a dry and more seasonal tropical climate. While species displacement was stable over the course of two years, the presence of both species generally appears to fluctuate in tandem in areas of coexistence. Aedes albopictus was always more frequently found and abundant regardless of location and climatic season. The heterogenous environmental conditions of Panama shape the competitive outcome and micro-geographic distribution of Aedes mosquitoes, with potential consequences for the transmission dynamics of urban and sylvatic zoonotic diseases.

     

Validation of Cut-Points for Evaluating the Intensity of Physical Activity with Accelerometry-Based Mean Amplitude Deviation (MAD)

Purpose

Our recent study of three accelerometer brands in various ambulatory activities showed that the mean amplitude deviation (MAD) of the resultant acceleration signal performed best in separating different intensity levels and provided excellent agreement between the three devices. The objective of this study was to derive a regression model that estimates oxygen consumption (VO2) from MAD values and validate the MAD-based cut-points for light, moderate and vigorous locomotion against VO₂ within a wide range of speeds.

Methods

29 participants performed a pace-conducted non-stop test on a 200 m long indoor track. The initial speed was 0.6 m/s and it was increased by 0.4 m/s every 2.5 minutes until volitional exhaustion. The participants could freely decide whether they preferred to walk or run. During the test they carried a hip-mounted tri-axial accelerometer and mobile metabolic analyzer. The MAD was calculated from the raw acceleration data and compared to directly measured incident VO₂. Cut-point between light and moderate activity was set to 3.0 metabolic equivalent (MET, 1 MET = 3.5 ml · kg^-1 · min^-1) and between moderate and vigorous activity to 6.0 MET as per standard use.

Results

The MAD and VO₂ showed a very strong association. Within individuals, the range of r values was from 0.927 to 0.991 providing the mean r = 0.969. The optimal MAD cut-point for 3.0 MET was 91 mg (milligravity) and 414 mg for 6.0 MET.

Conclusion

The present study showed that the MAD is a valid method in terms of the VO₂ within a wide range of ambulatory activities from slow walking to fast running. Being a device-independent trait, the MAD facilitates directly comparable, accurate results on the intensity of physical activity with all accelerometers providing tri-axial raw data.