The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation

A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.     

Displacement of the occluding loop by the parasite protein, chagasin, results in efficient inhibition of human cathepsin B

Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.     

A century of generalization

We review data from both ethology and psychology about generalization, that is how animals respond to sets of stimuli including familiar and novel stimuli. Our main conclusion is that patterns of generalization are largely independent of systematic group (evidence is available for insects, fish, amphibians, reptiles, birds and mammals, including humans), behavioural context (feeding, drinking, courting, etc.), sensory modality (light, sound, etc.) and of whether reaction to stimuli is learned or genetically inherited. These universalities suggest that generalization originates from general properties of nervous systems, and that evolutionary strategies to cope with novelty and variability in stimulation may be limited. Two major shapes of the generalization gradient can be identified, corresponding to two types of stimulus dimensions. When changes in stimulation involve a rearrangement of a constant amount of stimulation on the sense organs, the generalization gradient peaks close to familiar stimuli, and peak responding is not much higher than responding to familiar stimuli. Contrary to what is often claimed, such gradients are better described by Gaussian curves than by exponentials. When the stimulus dimension involves a variation in the intensity of stimulation, the gradient is often monotonic, and responding to some novel stimuli is considerably stronger than responding to familiar stimuli. Lastly, when several or many familiar stimuli are close to each other predictable biases in responding occur, along all studied dimensions. We do not find differences between biases referred to as peak shift and biases referred to as supernormal stimulation. We conclude by discussing theoretical issues.     

Regulated expression and intracellular localization of cystatin F in human U937 cells.

Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin ( approximately 25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3-4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPalpha, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all-trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong ( approximately 18-fold) down-regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.     

A small region of the natural killer cell receptor, Siglec-7, is responsible for its preferred binding to alpha 2,8-disialyl and branched alpha 2,6-sialyl residues. A comparison with Siglec-9.

Siglec-7 is a sialic acid-binding lectin recently identified as an inhibitory receptor on natural killer cells. Here we characterize the sugar-binding specificity of Siglec-7 expressed on Chinese hamster ovary cells using polyvalent streptavidin-based glyco-probes. Glyco-probes carrying unique oligosaccharide structures such as GD3 (NeuAc alpha 2,8NeuAc alpha 2,3Gal beta 1,4Glc) and LSTb (Gal beta 1,3[NeuAc alpha 2,6]GlcNAc beta 1,3Gal beta 1,4Glc) oligosaccharides bound to Siglec-7 better than those carrying LSTc (NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal beta 1,4Glc) or GD1a (NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,4[NeuAc alpha 2,3]Gal beta 1,4Glc) oligosaccharides. In contrast, Siglec-9, which is 84% identical to Siglec-7, did not bind to the GD3 and LSTb probes but did bind to the LSTc and GD1a probes. To identify a region(s) responsible for their difference in binding specificity, we prepared a series of V-set domain chimeras between Siglecs-7 and -9. Substitution of a small region, Asn(70)-Lys(75), of Siglec-7 with the equivalent region of Siglec-9 resulted in loss of Siglec-7-like binding specificity and acquisition of Siglec-9-like binding properties. In comparison, a Siglec-9-based chimera, which contains Asn(70)-Lys(75) with additional amino acids derived from Siglec-7, exhibited Siglec-7-like specificity. These results, combined with molecular modeling, suggest that the C-C' loop in the sugar-binding domain plays a major role in determining the binding specificities of Siglecs-7 and -9.     

Mapping of an immotile short tail sperm defect in the Finnish Yorkshire on porcine Chromosome 16

An immotile short tail sperm defect has recently been identified as a hereditary disorder present within the Finnish Yorkshire pig population. The syndrome is inherited as an autosomal recessive disease exclusively expressed in male individuals as shorter sperm tail length and immotile spermatozoa. Based on the assumption of a recent common origin of the disease-causing mutation, a genome-wide search was performed with 228 evenly spaced microsatellites by homozygosity mapping of affected and unaffected DNA pools. One locus, SW2411 on Chr 16, demonstrated a significantly skewed allele distribution between the two pools. Linkage analysis of five markers in this region mapped the disease-causing gene within a 6-cM confidence interval region with a highest LOD score of 7.7 at marker SW419. It appears that three-marker haplotypes can be used for marker-assisted selection within analyzed pedigrees. Furthermore, future fine mapping may reveal a more precise population-wide associated haplotype and facilitate identification of a new gene affecting sperm tail development. Received: 5 July 2001 / Accepted: 13 September 2001     

Raltegravir Cerebrospinal Fluid Concentrations in HIV-1 Infection

Introduction

Raltegravir is an HIV-1 integrase inhibitor currently used in treatment-experienced HIV-1-infected patients resistant to other drug classes. In order to assess its central nervous system penetration, we measured raltegravir concentrations in cerebrospinal fluid (CSF) and plasma in subjects receiving antiretroviral treatment regimens containing this drug.

Methods

Raltegravir concentrations were determined by liquid chromatography tandem mass spectrometry in 25 paired CSF and plasma samples from 16 HIV-1-infected individuals. The lower limit of quantitation was 2.0 ng/ml for CSF and 10 ng/ml for plasma.

Results

Twenty-four of the 25 CSF samples had detectable raltegravir concentrations with a median raltegravir concentration of 18.4 ng/ml (range, <2.0–126.0). The median plasma raltegravir concentration was 448 ng/ml (range, 37–5180). CSF raltegravir concentrations correlated with CSF:plasma albumin ratios and CSF albumin concentrations.

Conclusions

Approximately 50% of the CSF specimens exceeded the IC₉₅ levels reported to inhibit HIV-1 strains without resistance to integrase inhibitors. In addition to contributing to control of systemic HIV-1 infection, raltegravir achieves local inhibitory concentrations in CSF in most, but not all, patients. Blood-brain and blood-CSF barriers likely restrict drug entry, while enhanced permeability of these barriers enhances drug entry.     

Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce

Background  

Simultaneous saccharification and fermentation (SSF) is a promising process option for ethanol production from lignocellulosic materials. However, both the overall ethanol yield and the final ethanol concentration in the fermentation broth must be high. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content. A principal difficulty is to obtain an efficient pentose uptake in the presence of high glucose and inhibitor concentrations. Initial glucose present in pretreated spruce decreases the xylose utilization by yeast, due to competitive inhibition of sugar transport. In the current work, prefermentation was studied as a possible means to overcome the problem of competitive inhibition. The free hexoses, initially present in the slurry, were in these experiments fermented before adding the enzymes, thereby lowering the glucose concentration.     

Divergent apparent temperature sensitivity of terrestrial ecosystem respiration

Aims Recent studies revealed convergent temperature sensitivity of ecosystem respiration (R e) within aquatic ecosystems and between terrestrial and aquatic ecosystems. We do not know yet whether various terrestrial ecosystems have consistent or divergent temperature sensitivity. Here, we synthesized 163 eddy covariance flux sites across the world and examined the global variation of the apparent activation energy (Ea), which characterizes the apparent temperature sensitivity of and its interannual variability (IAV) as well as their controlling factors.Methods We used carbon fluxes and meteorological data across FLUXNET sites to calculate mean annual temperature, temperature range, precipitation, global radiation, potential radiation, gross primary productivity and R e by averaging the daily values over the years in each site. Furthermore, we analyzed the sites with>8 years data to examine the IAV of Ea and calculated the standard deviation of Ea across years at each site to characterize IAV.Important findings The results showed a widely global variation of Ea, with significantly lower values in the tropical and subtropical areas than in temperate and boreal areas, and significantly higher values in grasslands and wetlands than that in deciduous broadleaf forests and evergreen forests. Globally, spatial variations of Ea were explained by changes in temperature and an index of water availability with differing contribution of each explaining variable among climate zones and biomes. IAV and the corresponding coefficient of variation of Ea decreased with increasing latitude, but increased with radiation and corresponding mean annual temperature. The revealed patterns in the spatial and temporal variations of Ea and its controlling factors indicate divergent temperature sensitivity of R e, which could help to improve our predictive understanding of R e in response to climate change.