Automated analysis of vapor diffusion crystallization drops with an X-ray beam

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.     

Anti-CD20 Immunoglobulin G Radiolabeling with a 99mTc-Tricarbonyl Core: In Vitro and In Vivo Evaluations

In recent years, the diagnostic and therapeutic uses of radioisotopes have shown significant progress. Immunoglobulin (Ig) appears to be a promising tracer, particularly due to its ability to target selected antigens. The main objective of this study is to optimize and assess an Ig radiolabeling method with Technetium 99m (^99mTc), an attractive radioelement used widely for diagnostic imaging. Monoclonal anti-CD20 IgG was retained to study in vitro and in vivo radiolabeling impact. After IgG derivatization with 2-iminothiolane, IgG-SH was radiolabeled by an indirect method, using a ^99mTc-tricarbonyl core. Radiolabeling stability was evaluated over 24h by thin-layer chromatography. IgG integrity was checked by sodium dodecyl sulfate—polyacrylamide gel electrophoresis coupled with Western blot and autoradiography. The radiolabeled Ig’s immunoaffinity was assessed in vitro by a radioimmunoassay method and binding experiments with cells (EL4-hCD20 and EL4-WT). Biodistribution studies were performed in normal BALB/c mice. Tumor uptake was assessed in mice bearing EL4-hCD20 and EL4-WT subcutaneous xenografts. With optimized method, high radiolabeling yields were obtained (95.9 ± 3.5%). ^99mTc-IgG-SH was stable in phosphate-buffered saline (4°C and 25°C) and in serum (37°C), even if important sensitivity to transchelation was observed. IgG was not degraded by derivatization and radiolabeling, as shown by Western blot and autoradiography results. ^99mTc-anti-CD20 IgG-SH immunoaffinity was estimated with Kd = 35 nM by both methods. In vivo biodistribution studies for 48h showed significant accumulation of radioactivity in plasma, liver, spleen, lungs and kidneys. Planar scintigraphy of mice bearing tumors showed a significant uptake of ^99mTc-anti-CD20 IgG-SH in CD20⁺ tumor versus CD20^- tumor. Radiolabeling of derivatized IgG with ^99mTc-tricarbonyl was effective, stable and required few antibody amounts. This attractive radiolabeling method is “antibody safe” and preserves Ig affinity for antigen, as shown by both in vitro and in vivo experiments. This method could easily be used with noncommercial IgG or other antibody isotypes.     

Phylogenetic analysis of anostracans (Branchiopoda: Anostraca) inferred from nuclear 18S ribosomal DNA (18S rDNA) sequences

The nuclear small subunit ribosomal DNA (18S rDNA) of 27 anostracans (Branchiopoda: Anostraca) belonging to 14 genera and eight out of nine traditionally recognized families has been sequenced and used for phylogenetic analysis. The 18S rDNA phylogeny shows that the anostracans are monophyletic. The taxa under examination form two clades of subordinal level and eight clades of family level. Two families the Polyartemiidae and Linderiellidae are suppressed and merged with the Chirocephalidae, of which together they form a subfamily. In contrast, the Parartemiinae are removed from the Branchipodidae, raised to family level (Parartemiidae) and cluster as a sister group to the Artemiidae in a clade defined here as the Artemiina (new suborder). A number of morphological traits support this new suborder. The Branchipodidae are separated into two families, the Branchipodidae and Tanymastigidae (new family). The relationship between Dendrocephalus and Thamnocephalus requires further study and needs the addition of Branchinella sequences to decide whether the Thamnocephalidae are monophyletic. Surprisingly, Polyartemiella hazeni and Polyartemia forcipata ("Family" Polyartemiidae), with 17 and 19 thoracic segments and pairs of trunk limb as opposed to all other anostracans with only 11 pairs, do not cluster but are separated by Linderiella santarosae ("Family" Linderiellidae), which has 11 pairs of trunk limbs. All appear to be part of the Chirocephalidae and share one morphological character: double pre-epipodites on at least part of their legs. That Linderiella is part of the Polyartemiinae suggests that multiplication of the number of limbs occurred once, but was lost again in Linderiella. Within Chirocephalidae, we found two further clades, the Eubranchipus-Pristicephalus clade and the Chirocephalus clade. Pristicephalus is reinstated as a genus.     

Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing

Human mesenchymal stromal cells (MSC) can suppress T-cell activation in vitro in an indoleamine 2,3-dioxygenase (IDO)-dependent manner. However, their clinical effects on immune ailments have been inconsistent, with a recent phase III study showing no benefit in acute graft-versus-host disease (GvHD). We here tested the hypothesis that the banked, cryopreserved MSC often used in clinical trials display biologic properties distinct from that of MSC in the log phase of growth typically examined in pre-clinical studies. In freshly thawed cryopreserved MSC derived from normal human volunteers, we observed that MSC up-regulate heat-shock proteins, are refractory to interferon (IFN)-γ-induced up-regulation of IDO, and are compromised in suppressing CD3/CD28-driven T cell proliferation. Immune suppressor activity, IFN-γ responsiveness and induction of IDO were fully restored following 24 h of MSC tissue culture post-thaw. These results highlight a possible cause for the inefficacy of MSC-based immunotherapy reported in clinical trials using cryopreserved MSC thawed immediately prior to infusion.     

Identification of the domain in the human interleukin-11 receptor that mediates ligand binding

The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily. The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130. The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3. The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3). We investigated the structural and functional role of the third extracellular receptor domain of IL-11R. A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding. Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11. To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified. For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used. By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.     

Drosophila melanogaster Acetyl-CoA-Carboxylase Sustains a Fatty Acid-Dependent Remote Signal to Waterproof the Respiratory System

Fatty acid (FA) metabolism plays a central role in body homeostasis and related diseases. Thus, FA metabolic enzymes are attractive targets for drug therapy. Mouse studies on Acetyl-coenzymeA-carboxylase (ACC), the rate-limiting enzyme for FA synthesis, have highlighted its homeostatic role in liver and adipose tissue. We took advantage of the powerful genetics of Drosophila melanogaster to investigate the role of the unique Drosophila ACC homologue in the fat body and the oenocytes. The fat body accomplishes hepatic and storage functions, whereas the oenocytes are proposed to produce the cuticular lipids and to contribute to the hepatic function. RNA-interfering disruption of ACC in the fat body does not affect viability but does result in a dramatic reduction in triglyceride storage and a concurrent increase in glycogen accumulation. These metabolic perturbations further highlight the role of triglyceride and glycogen storage in controlling circulatory sugar levels, thereby validating Drosophila as a relevant model to explore the tissue-specific function of FA metabolic enzymes. In contrast, ACC disruption in the oenocytes through RNA-interference or tissue-targeted mutation induces lethality, as does oenocyte ablation. Surprisingly, this lethality is associated with a failure in the watertightness of the spiracles-the organs controlling the entry of air into the trachea. At the cellular level, we have observed that, in defective spiracles, lipids fail to transfer from the spiracular gland to the point of air entry. This phenotype is caused by disrupted synthesis of a putative very-long-chain-FA (VLCFA) within the oenocytes, which ultimately results in a lethal anoxic issue. Preventing liquid entry into respiratory systems is a universal issue for air-breathing animals. Here, we have shown that, in Drosophila, this process is controlled by a putative VLCFA produced within the oenocytes.     

Effects of Beta-Alanine Supplementation on Brain Homocarnosine/Carnosine Signal and Cognitive Function: An Exploratory Study

Objectives

Two independent studies were conducted to examine the effects of 28 d of beta-alanine supplementation at 6.4 g d^-1 on brain homocarnosine/carnosine signal in omnivores and vegetarians (Study 1) and on cognitive function before and after exercise in trained cyclists (Study 2).

Methods

In Study 1, seven healthy vegetarians (3 women and 4 men) and seven age- and sex-matched omnivores undertook a brain 1H-MRS exam at baseline and after beta-alanine supplementation. In study 2, nineteen trained male cyclists completed four 20-Km cycling time trials (two pre supplementation and two post supplementation), with a battery of cognitive function tests (Stroop test, Sternberg paradigm, Rapid Visual Information Processing task) being performed before and after exercise on each occasion.

Results

In Study 1, there were no within-group effects of beta-alanine supplementation on brain homocarnosine/carnosine signal in either vegetarians (p = 0.99) or omnivores (p = 0.27); nor was there any effect when data from both groups were pooled (p = 0.19). Similarly, there was no group by time interaction for brain homocarnosine/carnosine signal (p = 0.27). In study 2, exercise improved cognitive function across all tests (P<0.05), although there was no effect (P>0.05) of beta-alanine supplementation on response times or accuracy for the Stroop test, Sternberg paradigm or RVIP task at rest or after exercise.

Conclusion

28 d of beta-alanine supplementation at 6.4g d^-1 appeared not to influence brain homocarnosine/carnosine signal in either omnivores or vegetarians; nor did it influence cognitive function before or after exercise in trained cyclists.     

Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Exploration of the diaphorase activity of neutrophil NADPH oxidase.

In the O2- generating flavocytochrome b, the membrane-bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPH --> FAD --> heme b -->O2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by INT as efficiently as by O2 even in the presence of concentrations of 5-deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite-reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite-reduced hemin. Third, INT competitively inhibited the O2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.