Towards unambiguous assignment of methyl-containing residues by double and triple sensitivity-enhanced HCCmHm-TOCSY experiments

Chemical shift assignment of methyl-containing residues is essential in protein NMR spectroscopy, as these residues are abundant in protein interiors and provide the vast majority of long-range NOE connectivities for structure determination. These residues also constitute an integral part of hydrophobic cavities, the surroundings for many enzymatic reactions. Here we present a powerful strategy for the assignment of methyl-containing residues in a uniformly ¹³C/¹⁵N double labeled protein sample. The approach is based on novel four-dimensional HCCmHm-TOCSY experiments, two of them utilizing gradient selection and sensitivity enhancement in all three indirectly detected dimensions. Regardless of the number of dimensions, the proposed experiments can be executed using only one transient per FID, providing outstanding resolution and sensitivity. A complete assignment of the 51 methyl-containing residues in the 16 kDa Mus musculus coactosin was accomplished using a four-dimensional HCCmHm-TOCSY spectrum recorded in 16 hours.     

A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia

Following neuronal injury, microglia initiate repair by phagocytosing dead neurons without eliciting inflammation. Prior evidence indicates triggering receptor expressed by myeloid cells-2 (TREM2) promotes phagocytosis and retards inflammation. However, evidence that microglia and neurons directly interact through TREM2 to orchestrate microglial function is lacking. We here demonstrate that TREM2 interacts with endogenous ligands on neurons. Staining with TREM2-Fc identified TREM2 ligands (TREM2-L) on Neuro2A cells and on cultured cortical and dopamine neurons. Apoptosis greatly increased the expression of TREM2-L. Furthermore, apoptotic neurons stimulated TREM2 signaling, and an anti-TREM2 mAb blocked stimulation. To examine the interaction between TREM2 and TREM2-L in phagocytosis, we studied BV2 microglial cells and their engulfment of apoptotic Neuro2A. One of our anti-TREM2 mAb, but not others, reduced engulfment, suggesting the presence of a functional site on TREM2 interacting with neurons. Further, Chinese hamster ovary cells transfected with TREM2 conferred phagocytic activity of neuronal cells demonstrating that TREM2 is both required and sufficient for competent uptake of apoptotic neuronal cells. Finally, while TREM2-L are expressed on neurons, TREM2 is not; in the brain, it is found on microglia. TREM2 and TREM2-L form a receptor–ligand pair connecting microglia with apoptotic neurons, directing removal of damaged cells to allow repair.     

Interactive effect of elevated temperature and O<Subscript>3</Subscript> on antioxidant capacity and gas exchange in <Emphasis Type="Italic">Betula pendula</Emphasis> saplings

We studied the effects of slightly elevated temperature (T), O₃ concentration (O₃) and their combination (T + O₃) on the antioxidant defense, gas exchange and total leaf area of Betula pendula saplings in field conditions. During the second year of the experiment, T enhanced the total leaf area, net photosynthesis (P _n) and maximum capacity of carboxylation, redox state of ascorbate and total antioxidant capacity in the apoplast. O₃ did not affect the total leaf area, but P _n was slightly and g _s significantly reduced. The saplings responded to elevated O₃ level by closing the stomata and by developing leaves with a lower leaf area per mass, rather than by accumulating ascorbate in the apoplast. The effects of T and O₃ on total leaf area and P _n were counteractive. Elevated O₃ reduced the saplings’ ability to utilize the warmer growth environment by increasing the stomatal limitation for photosynthesis and by reducing the redox state of ascorbate in the apoplast in the combination treatment as compared to T alone.     

Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

Background

Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated.

Results

Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material.

Conclusion

Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.     

Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide

Retroanandamide (2f) and its 10 analogues (1a-e, 2a-e) were synthesized and evaluated for the cannabinoid receptor activation by a [35S]GTPgammaS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was explored by synthesizing retroanandamide analogues having different chain lengths (1a-e, C19; 2a-f, C20). All the synthesized compounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.     

Insulin sensitivity regulates cholesterol metabolism to a greater extent than obesity: lessons from the METSIM Study

Cholesterol synthesis is upregulated and absorption downregulated in insulin resistance and in type 2 dia-betes. We investigated whether alterations in cholesterol metabolism are observed across the glucose tolerance status, from normoglycemia through impaired glucose tolerance to type 2 diabetes, in 781 randomly selected men 45 to 70 years of age from a population-based Metabolic Syndrome in Men Study. Cholesterol metabolism was assayed using surrogate serum markers, squalene, and noncholesterol sterols. The study population was classified into subgroups according to glucose tolerance as follows: normoglycemia, impaired fasting glucose, impaired glucose tolerance, and type 2 diabetes. LDL cholesterol did not differ between the groups. Cholesterol synthesis markers were lowest and absorption markers highest in normoglycemia. Sitosterol was lower in subjects with impaired fasting glucose compared with normoglycemic subjects (113 ± 7 vs. 136 ± 3 10² μmol/mmol of cholesterol, P < 0.05). LDL cholesterol was not associated with lathosterol/sitosterol ratio, a marker of cholesterol metabolism. Peripheral insulin sensitivity evaluated by the Matsuda index was associated with the lathosterol/sitosterol ratio in the entire population (r = −0.457, P < 0.001) and with that of lathosterol/cholestanol independently of obesity. In conclusion, cholesterol metabolism was altered already from subjects with impaired fasting glucose. Upregulated cholesterol synthesis was associated with peripheral insulin resistance independent of obesity.     

High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma

We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography–tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005–5 μmol/L. The extraction recoveries for all the analytes fell in the range of 88–101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6 h at room temperature, at least three freeze–thaw cycles, in the −70 °C or −20 °C freezer for 2 months, and also in the reconstitution solution at 8 °C for 48 h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.     

Crystal structure of aclacinomycin-10-hydroxylase, a S-adenosyl-L-methionine-dependent methyltransferase homolog involved in anthracycline biosynthesis in Streptomyces purpurascens

Anthracyclines are aromatic polyketide antibiotics, and several of these compounds are widely used as anti-tumor drugs in chemotherapy. Aclacinomycin-10-hydroxylase (RdmB) is one of the tailoring enzymes that modify the polyketide backbone in the biosynthesis of these metabolites. RdmB, a S-adenosyl-L-methionine-dependent methyltransferase homolog, catalyses the hydroxylation of 15-demethoxy-epsilon-rhodomycin to beta-rhodomycin, one step in rhodomycin biosynthesis in Streptomyces purpurascens. The crystal structure of RdmB, determined by multiwavelength anomalous diffraction to 2.1A resolution, reveals that the enzyme subunit has a fold similar to methyltransferases and binds S-adenosyl-L-methionine. The N-terminal domain, which consists almost exclusively of alpha-helices, is involved in dimerization. The C-terminal domain contains a typical alpha/beta nucleotide-binding fold, which binds S-adenosyl-L-methionine, and several of the residues interacting with the cofactor are conserved in O-methyltransferases. Adjacent to the S-adenosyl-L-methionine molecule there is a large cleft extending to the enzyme surface of sufficient size to bind the substrate. Analysis of the putative substrate-binding pocket suggests that there is no enzymatic group in proximity of the substrate 15-demethoxy-epsilon-rhodomycin, which could assist in proton abstraction and thus facilitate methyl transfer. The lack of a suitably positioned catalytic base might thus be one of the features responsible for the inability of the enzyme to act as a methyltransferase.     

Confirmation of Escherichia coli and its distinction from Klebsiella species by gas and indole formation at 44 and 44.5 degrees C

AIMS: In the enumeration of coliform bacteria, confirmation of Escherichia coli has been based upon gas and indole production at the elevated incubation temperature. The test for gas production has recently been questioned. The aim of this study was to investigate the impact of gas production test on the reliability of confirmation of E. coli. METHODS AND RESULTS: The impact of several media on growth, gas and/or indole formation was tested at 44 and 44.5 degrees C using 547 environmental isolates. These were mainly E. coli, Klebsiella pneumoniae, K. oxytoca and Enterobacter cloacae strains. Another set of 250 faecal and environmental klebsiellae were tested for their maximum temperature for growth (Tmax) and for gas formation. Escherichia coli and even K. pneumoniae grew well in all the media, but gas production was more dependent on the medium used. Growth of the mainly gas negative Ent. cloacae and K. oxytoca strains was still more sensitive to the medium and incubation conditions. Tryptophan salt broth was the most productive medium for the indole test, followed by lauryl tryptose mannitole and tryptone mannitol ricinoleate broth (TRM). Tmax of K. oxytoca was clearly lower than Tmax of K. pneumoniae but a rather high fraction of its isolates produced indole at 44.5 degrees C. CONCLUSIONS: False-positive E. coli confirmation is possible if gas production is not tested for and the confirmation is based on indole test only. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous positive results on routine analysis for E. coli can occur.     

Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains

A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.