Autolysis loop restricts the specificity of activated protein C: analysis by FRET and functional assays

We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S. The activity of APC-Tryp 143-154 toward fVa was identical to that of wild-type APC both in the presence and absence of protein S. However, the reactivity of APC-Tryp 143-154 with plasma inhibitors was preferentially improved independent of protein S. The FRET analysis revealed a dramatic change in the active-site topography of APC both in the absence and presence of protein S. Anisotropy measurements revealed that the fluorescein dye has a remarkable degree of rotational freedom in the active-site of APC-Tryp 143-154. These results suggest that the autolysis loop of APC may not be a target for modulation by protein S. This loop, however, plays a critical role in restricting both the specificity and spatial environment of the active-site groove of APC.     

Real-time treatment of dairy manure: implications of oxidation reduction potential regimes to nutrient management strategies

A pilot-scale sequencing batch reactor (SBR) was operated at a dairy farm to test real-time based control in winter operation conditions. A combination of high loading and low oxidation reduction potential (ORP) conditions in the aerobic stage of SBR treatment (an end value of -50 to -150 mV) inhibited nitrification while maintaining carbon removal. After a period of over-aeration over several cycles, the ORP at the end of the aerobic stage increased to values of 50-75 mV. Subsequently, nitrification was observed, accompanied by higher total cycle times. Significant increase in removal efficiencies of ammonical nitrogen (alpha<0.0001) and chemical oxygen demand (alpha<0.001) were observed for the high ORP phase. It is postulated that higher ORP regimes are needed for nitrification. In low ORP regimes, nitrification is absent or occurs at an extremely low rate. It is also noted that nitrifying systems treating high strength animal manure can possibly lead to unacceptably high levels of effluent nitrate+nitrite nitrogen (NO(x)-N). Two manure management schemes are proposed that give the farmer an option to either retain the nutrients, or remove them from the wastewater. Some advantages and disadvantages of the schemes are also discussed.     

Effects of acidosis on ventricular myocyte shortening and intracellular Ca2+ in streptozotocin-induced diabetic rats

We have investigated the effects of acute acidosis on ventricular myocyte shortening and intracellular Ca²⁺ in streptozotocin (STZ)-induced diabetic rat. Shortening and intracellular Ca²⁺ were measured in electrically stimulated myocytes superfused with either normal Tyrode solution pH adjusted to either 7.4 (control solution) or 6.4 (acid solution). Experiments were performed at 35–36°C. At 8–12 weeks after treatment, the rats that received STZ had lower body and heart weights compared to controls, and blood glucose was characteristically increased. Contractile defects in myocytes from diabetic rat were characterized by prolonged time to peak shortening. Superfusion of myocytes from control and diabetic rats with acid solution caused a significant reduction in the amplitude of shortening; however, the magnitude of the response was not altered by STZ treatment. Acid solution also caused significant and quantitatively similar reductions in the amplitude of Ca²⁺ transients in myocytes from control and diabetic rats. Effects of acute acidosis on amplitude of myocyte contraction and Ca²⁺ transient were not significantly altered by STZ treatment. Altered myofilament sensitivity to Ca²⁺ and altered mechanisms of sarcoplasmic reticulum Ca²⁺ transport might partly underlie the acidosis-evoked reduction in amplitude of shortening in myocytes from control and STZ-induced diabetic rat. (Mol Cell Biochem 261: 227–233, 2004)     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na⁺-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na⁺-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K_m=18.7 μM; V_max=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K_m=11.5 μM and V_max=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na⁺-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na⁺-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Unidirectional movement of tracers along the stolon of Saxifraga sarmentosa

Summary When radioactive tracer is applied locally to the stolon of Saxifraga its long-distance movement after 18 hours is found to be strongly polarised; there is in addition a short-distance movement which is unpolarised. With caesium, the long-distance movement is predominantly in the phloem; with strontium in the xylem. These interpretations, a priori probable, were confirmed by artifically reversing, separately, the xylem and the phloem currents. With long pieces of excised stolon only the unpolarised short-distance movement is observed. These results constitute evidence against simultaneous bidirectional translocation in the same sieve tube, and are consistent with either the Münch or the electro-osmotic theory.     

DNA photolyase of enterococci: possible explanation for its low sunlight inactivation rate

DNA photolyase is perhaps the most ancient and direct arsenal in curing the UV-induced dimers formed in the microbial genome. Out of two cofactors of the enzyme, catalytic and light harvesting, differences in the latter have provided basis for categorizing photolyases of prokaryotes as folate and deazaflavin types. In the present study, the homology modeling of DNA photolyase of Enterococcus faecalis was undertaken. The predicted models were structurally compared with the crystal structure coordinates of photolyases from Escherichia coli (folate type) and Anacystis nidulans (deazaflavin type). Discrepancies present in the multiple sequence alignment and tertiary structures, particularly at the light harvesting cofactor (methenyltetrahydrofolic acid, MTHF; 8-hydroxy-5-deazaflavin, 8-HDF) binding sites indicated the mechanistic nature of enterococcal photolyase. Concisely, despite the greater holistic homology with folate-type photolyase, enterococcal photolyase was characterized as deazaflavin-type. The presence of 8-HDF binding sites and groove architecture of substrate binding sites were also found supportive in this regard. The inter cofactor distance and/or orientation also implied to the efficient energy transfer in photolyase of Enterococcus in comparison with E. coli. In addition, we observed relatively high protein deformability in the enterococcal genome, which may favors the repair action of photolyase. The findings are expected to provide molecular insights into the difference in sunlight inactivation rate of two important fecal contamination indicators, namely Enterococcus and E. coli.     

Influenza virus replication in human cells exposed to the pesticide emulsifier Atlox

The emulsifier Atlox was capable of enhancing the infectivity of influenza virus in Chang human conjunctiva cells. Prior to infection, pre-treatment of cells with 2.5 to 10 ppm of Atlox for 6 to 8 h was necessary to detect an increase in virus production. Although there was no difference in the amount of adsorbed virus, the number of successfully infected cells was at least 1 log higher in Atlox treated cells as compared to the controls. Monitoring the 51Cr release from Atlox treated cells indicated a temporary change in membrane structure of the cells as one of the mechanisms of virus enhancement.     

Predicting the Distribution Pattern of Small Carnivores in Response to Environmental Factors in the Western Ghats

Due to their secretive habits, predicting the pattern of spatial distribution of small carnivores has been typically challenging, yet for conservation management it is essential to understand the association between this group of animals and environmental factors. We applied maximum entropy modeling (MaxEnt) to build distribution models and identify environmental predictors including bioclimatic variables, forest and land cover type, topography, vegetation index and anthropogenic variables for six small carnivore species in Mudumalai Tiger Reserve. Species occurrence records were collated from camera-traps and vehicle transects during the years 2010 and 2011. We used the average training gain from forty model runs for each species to select the best set of predictors. The area under the curve (AUC) of the receiver operating characteristic plot (ROC) ranged from 0.81 to 0.93 for the training data and 0.72 to 0.87 for the test data. In habitat models for F. chaus, P. hermaphroditus, and H. smithii “distance to village” and precipitation of the warmest quarter emerged as some of the most important variables. “Distance to village” and aspect were important for V. indica while “distance to village” and precipitation of the coldest quarter were significant for H. vitticollis. “Distance to village”, precipitation of the warmest quarter and land cover were influential variables in the distribution of H. edwardsii. The map of predicted probabilities of occurrence showed potentially suitable habitats accounting for 46 km² of the reserve for F. chaus, 62 km² for V. indica, 30 km² for P. hermaphroditus, 63 km² for H. vitticollis, 45 km² for H. smithii and 28 km² for H. edwardsii. Habitat heterogeneity driven by the east-west climatic gradient was correlated with the spatial distribution of small carnivores. This study exemplifies the usefulness of modeling small carnivore distribution to prioritize and direct conservation planning for habitat specialists in southern India.     

α-Synuclein Levels in Blood Plasma from LRRK2 Mutation Carriers

The diagnosis of Parkinson’s disease (PD) remains primarily a clinical issue, based mainly on phenotypic patterns. The identification of biomarkers capable of permitting the preclinical detection of PD is critically needed. α-Synuclein is a key protein in PD, with missense and multiplication mutations in the gene encoding α-synuclein (SNCA) having been reported in familial cases of PD, and accumulation of the protein identified in Lewy bodies (LBs) and Lewy neurites (LNs) in affected brain regions. With the objective of validating the use of α-synuclein as a clinical or progressive biomarker in an accessible tissue, we used an enzyme-linked immunosorbent assay (ELISA) to measure α-synuclein levels in the peripheral blood plasma of idiopathic PD and LRRK2 mutation carrier patients and compared our findings with healthy control subjects. Compared to healthy controls, we found a significant decrease in plasma total α-synuclein levels in idiopathic PD (iPD) patients (n = 134, p = 0.010). However, the reduction was less significant in patients who were LRRK2 mutation carriers (n = 32, p = 0.133). This lack of significance could be due to the small number of individuals employed in this group. No predictive value of total α-synuclein in the diagnosis of PD was found in a receiver operating characteristic (ROC) curve analysis. Although this is a pilot study requiring corroboration on a larger cohort of patients, our results highlight the possible use of plasma α-synuclein as a biomarker for PD.