Measurement of Ice Nucleation-Active Bacteria on Plants and in Precipitation by Quantitative PCR

Ice nucleation-active (INA) bacteria may function as high-temperature ice-nucleating particles (INP) in clouds, but their effective contribution to atmospheric processes, i.e., their potential to trigger glaciation and precipitation, remains uncertain. We know little about their abundance on natural vegetation, factors that trigger their release, or persistence of their ice nucleation activity once airborne. To facilitate these investigations, we developed two quantitative PCR (qPCR) tests of the ina gene to directly count INA bacteria in environmental samples. Each of two primer pairs amplified most alleles of the ina gene and, taken together, they should amplify all known alleles. To aid primer design, we collected many new INA isolates. Alignment of their partial ina sequences revealed new and deeply branching clades, including sequences from Pseudomonas syringae pv. atropurpurea, Ps. viridiflava, Pantoea agglomerans, Xanthomonas campestris, and possibly Ps. putida, Ps. auricularis, and Ps. poae. qPCR of leaf washings recorded ∼10⁸ina genes g⁻¹ fresh weight of foliage on cereals and 10⁵ to 10⁷ g⁻¹ on broadleaf crops. Much lower populations were found on most naturally occurring vegetation. In fresh snow, ina genes from various INA bacteria were detected in about half the samples but at abundances that could have accounted for only a minor proportion of INP at −10°C (assuming one ina gene per INA bacterium). Despite this, an apparent biological source contributed an average of ∼85% of INP active at −10°C in snow samples. In contrast, a thunderstorm hail sample contained 0.3 INA bacteria per INP active at −10°C, suggesting a significant contribution to this sample.     

Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.     

Arene- and quinoline-sulfonamides as novel 5-HT7 receptor ligands

Novel arene- and quinolinesulfonamides were synthesized using different solutions and a solid-support methodology, and were evaluated for their affinity for 5-HT(1A), 5-HT(2A), 5-HT(6), and 5-HT(7) receptors. Compound 54 (N-Ethyl-N-[4-(1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinolin-2-yl)butyl]-8-quinolinesulfonamide) was identified as potent 5-HT(7) antagonist (K(i)=13 nM, K(B)=140 nM) with good selectivity over 5-HT(1A), 5-HT(2A), 5-HT(6) receptors. In the FST in mice, it reduced immobility in a manner similar to the selective 5-HT(7) antagonist SB-269970.     

Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the beta-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.     

Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.     

Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase.

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.     

Coordination conjugates of therapeutic proteins with drug carriers: a new approach for versatile advanced drug delivery

We present here a general system for the coordination attachment of therapeutic proteins to a drug delivery system and its application in combined therapy. Proof of concept is demonstrated by the synthesis and testing of the targeted drug delivery system for cytostatics, which is based on a combination of the drug carrier Zn-porphyrin-cyclodextrin conjugates and their supramolecular coordination complexes with immunoglobulins. This system can be as readily used for a variety of therapeutic and targeting proteins including PAs, MAs, lectins, and HSA. Moreover, it allows combined photodynamic therapy, cell targeted chemotherapy and immunotherapy. When tested in a mouse model with human C32 carcinoma, the therapeutic superiority of the coordination assembly nanosystem was shown in comparison with the efficacy of building blocks used for the construction of the system.     

Angiopoietin-like protein 4: development, analytical characterization, and clinical testing of a new ELISA

The aim of our work was to develop an assay for the determination of angiopoietin-like protein 4 (Angplt4) in human blood, and to investigate its levels in healthy volunteers and donors suffer from metabolic syndrome. We developed and evaluated the sandwich ELISA method for the quantitative determination of human Angplt4 in serum samples. We conducted also the pilot study on individuals with metabolic syndrome or familiar hypercholesterolemia and healthy probands and measured blood pressure, waist circumference, Angplt4 serum levels, serum cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, insulin, glucose, A-FABP and calculate BMI and QUICKI insulin sensitivity index. In the study on 30 healthy volunteers we demonstrated that sex or age is not the determinant for Angplt4 serum values. Furthermore, we tested 115 individuals with metabolic syndrome and found that probands with metabolic syndrome did not differ in Angplt4 values than healthy individuals from the first study (medians 8.7 vs. 8.1 ng/ml, p = 0.6). Individuals with metabolic syndrome did not differ in sex or age from healthy. Angplt4 values correlated with the HDL-cholesterol (r = -0.25; p < 0.01), FGF-21 (r = 0.23, p < 0.01), glucose (r = 0.17; p = 0.03), uric acid (r = 0.17; p = 0.49), lipocalin-2 (r = 0.23, p < 0.01), triacylglycerols (r = 0.25; p < 0.01) and number or characters of metabolic syndrome (r = 0.21; p < 0.01). No significant correlation was found between serum Angplt4 and BMI, WC or QUICKI. However, we performed stepwise regression and we found that Angplt4 was not an independent marker for metabolic syndrome. The patients from the metabolic syndrome group suffering diabetes mellitus (n = 83) did not differ in serum Angplt4 from the group of healthy patients, too. The pilot study supports the hypothesis about the role of Angplt4 as a new class of lipid metabolism modulator. Their values could be a new key predictors of metabolic syndrome. Further research is necessary to confirm our findings in individuals with dyslipidemia, obesity, coronary artery diseases and different medication in order to assess Angplt4 value as a risk predictor of accelerated atherosclerosis.