Vitek 2 ANC card versus BBL Crystal Anaerobe and RapID ANA II for identification of clinical anaerobic bacteria

The Vitek 2 Anaerobe and Corynebacterium Identification Card (ANC) was recently evaluated in a multicentre study. In the present work, this system was compared with the BBL Crystal Anaerobe and RapID ANA II panels. These kits were tested using 196 strains of anaerobes that had been previously identified by gas–liquid chromatography. Identification to the species or to the genus level was 75.0%, 81.1% and 70.9% for Crystal, RapID and Vitek, respectively. Vitek ANC failed to provide any identification in 20.4% of the strains, but it had fewer misidentifications than RapID. The confidence factors provided on the results report of each kit were not always correlated with a lower risk of major errors, with the exception of Vitek 2 in which a confidence factor higher than 0.86 excluded the risk of misidentification in more than 87% of isolates. The lower rate of identification by the Vitek and Crystal panels is mostly due the lower ability of these systems to identify the Clostridia. Overall, the three panels are comparable but need improvement to a better accuracy.     

Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa

Human leukocyte receptor IIIa (hFcγRIIIa) plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. In previous studies, a major impact of the presence of carbohydrates at Asn-162 on the binding between the receptor and the Fc part of wild type fucosylated or glycoengineered nonfucosylated antibodies has been shown. In this study, we performed a site directed carbohydrate analysis at hFcγRIIIa derived from human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells, respectively. Using mass spectrometry (MS) and a multienzyme protein digest, we analyzed the proteolysis-generated glycopeptides in detail. We could show that hFcγRIIIa expressed by HEK cells was mostly bearing multifucosylated biantennary Asn162-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could demonstrate that the glycan antennae with terminal GalNAc could be sialylated as indicated by a novel reporter ion HexNAcHexNAcNeuAc(+) (m/z 698.28) using a source induced dissociation (SID) scan in the MS cycle. In contrast to the hFcγRIIIa Asn-162 glycosylation pattern from HEK cells, the CHO cells derived receptor contains bi- and triantennary galactosylated and highly sialylated carbohydrates. Our data suggest that the type of expression host system was a dominating factor for formation of distinct glycopatterns of hFcγRIIIa, while the protein sequence and the site of glycosylation remained unchanged for both types of cells. Using surface plasmon resonance (SPR) interaction analysis, we show that the cell type and site specific glycosylation pattern of hFcγRIIIa influences its binding behavior to immunoglobulin molecules.     

Growth requirement for N as a criterion to assess the effects of physical manipulation on nitrate uptake fluxes in spinach

The effects of physical manipulation of hydroponically grown plants of spinach (Spinacia oleracea L., cvs Subito and Glares) on nitrate uptake fluxes were studied in a long-term experiment (3 days), and in short-term label experiments (2 h) with ¹³N-nitrate and ¹⁵N-nitrate. In the long-term experiment, net nitrate uptake rate (NNUR) was measured by following the nitrate depletion in the uptake solution, which was replaced at regular intervals. In the short-term experiments, NNUR and nitrate influx were measured by simultaneous application of ¹³N-nitrate and ¹⁵N-nitrate. Plants were gently transferred into the labelled uptake solution, as is usually done in nutrient uptake studies. In addition, a more severe physical manipulation was carried out, including blotting of the roots, to mimic pretreatments which involve more handling of the plants prior to uptake measurements. Nitrate influx was measured immediately after physical manipulation and after 2 h of recovery. To assess the impact of the physical manipulation the experimentally determined nitrate uptake fluxes were compared with the N demand for growth, defined as relative growth rate (RGR) times plant nitrogen concentration (PNC) of parallel plants, which were left undisturbed. Nitrate influx and efflux were both subject to changes after physical manipulation of the plants. Physical handling, however, did not always result in an alteration of NNUR, which complicates the determination of the length of the recovery period. The impact of the handling and the time course of the recovery depended on the severity of the disturbance and were independent of the light conditions during the experiments. Even after a gentle transfer of the plants, recovery, in most cases, was not complete within 2 h. The data emphasise the need for minimal disturbance of plants during the last hours prior to nutrient uptake measurements.     

Enhancement of antidepressant potency by a potentiator of AMPA receptors

1. AMPA receptor potentiators (ARPs) exhibit antidepressant-like activity in preclinical tests (for example, the forced swim test) that are highly predictive of efficacy in humans. Unlike most currently used antidepressants, ARPs do not elevate extracellular levels of biogenic amines (e.g., 5HT, NE) in prefrontal cortex at doses that are active in the forced swim test.2. The present series of experiments examined the effects of combining the ARP, LY 392098, with biogenic amine-based antidepressants in the forced swim test. Male, NIH Swiss mice were placed in a cylinder of water and observed for attempted escape behaviors and immobility.3. LY 392098 dose-dependently decreased immobility as did a range of classical antidepressants. At doses of LY 392098 below those that decreased immobility, this compound significantly increased the potency with which fluoxetine and citalopram (SSRI antidepressants), imipramine (tricyclic antidepressant), duoxetine (norepinephrine/serotonin uptake blocker), nisoxetine (norepinephrine uptake inhibitor), and rolipram (PDE4 inhibitor) decreased immobility in the forced swim test with potency shifts upward of 5-fold (fluoxetine, imipramine, and rolipram). Likewise, ineffective doses of the traditional antidepressants potentiated the effects LY 392098 with shifts in the dose-effect functions that were 10-fold or more for citalopram, fluoxetine, imipramine, and duloxetine.4. Combined with other evidence for a role of AMPA receptors in the efficacy of antidepressants, the current data suggest that the addition of an ARP may augment the activity and perhaps the onset of the therapeutic effects of biogenic amine and second messenger-based antidepressants.     

Molecular modeling of glycosyltransferases involved in the biosynthesis of blood group A,blood group B,Forssman, and iGb3 antigens and their interaction with substrates

A terminal alpha1-3 linked Gal or GalNAc sugar residue is the common structure found in several oligosaccharide antigens, such as blood groups A and B, the xeno-antigen, the Forssman antigen, and the isogloboside 3 (iGb3) glycolipid. The enzymes involved in the addition of this residue display strong amino acid sequence similarities, suggesting a common fold. From a recently solved crystal structure of the bovine alpha3-galactosyltransferase complexed with UDP, homology modeling methods were used to build the four other enzymes of this family in their locked conformation. Nucleotide-sugars, the Mn2+ ion, and oligosaccharide acceptors were docked in the models. Nine different amino acid regions are involved in the substrate binding sites. After geometry optimization of the complexes and analysis of the predicted structures, the basis of the specificities can be rationalized. In the nucleotide-sugar binding site, the specificity between Gal or GalNAc transferase activity is due to the relative size of two clue amino acids. In the acceptor site, the presence of up to three tryptophan residues define the complexity of the oligosaccharide that can be specifically recognized. The modeling study helps in rationalizing the crystallographic data obtained in this family and provides insights on the basis of substrate and donor recognition.     

Effects of deletion of the prolactin receptor on ovarian gene expression

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, and is mainly considered as a regulator of reproduction and cell growth. Null mutation of the PRL receptor (R) gene leads to female sterility due to a complete failure of embryo implantation. Pre-implantatory egg development, implantation and decidualization in the mouse appear to be dependent on ovarian rather than uterine PRLR expression, since progesterone replacement permits the rescue of normal implantation and early pregnancy. To better understand PRL receptor deficiency, we analyzed in detail ovarian and corpora lutea development of PRLR-/- females. The present study demonstrates that the ovulation rate is not different between PRLR+/+ and PRLR-/- mice. The corpus luteum is formed but an elevated level of apoptosis and extensive inhibition of angiogenesis occur during the luteal transition in the absence of prolactin signaling. These modifications lead to the decrease of LH receptor expression and consequently to a loss of the enzymatic cascades necessary to produce adequate levels of progesterone which are required for the maintenance of pregnancy.