Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions

Background  

To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of thesegenes.     

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Background  

Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).     

D-1-Nal4]endomorphin-2 is a potent micro-opioid receptor antagonist in the aequorin luminescence-based calcium assay

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca(2+) levels, was used to examine relative potency and efficacy of the micro-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) analogues containing D-3-(1-naphthyl)-alanine (D-1-Nal) or D-3-(2-naphthyl)-alanine (D-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their micro-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the micro-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with D-1-Nal or D-2-Nal substitutions in position 4 of endomorphin-2 are strong micro-opioid receptor antagonists, while those substituted in position 3 are partial agonists. Exceptional antagonist potency in the calcium assay was observed for [D-1-Nal(4)]endomorphin-2. The pA(2) value for this analogue was 7.95, compared to the value of 8.68 obtained for the universal, non-selective opioid antagonist of the alkaloid structure, naloxone. The obtained results were compared with the data from the hot-plate test in mice. In that in vivo assay [D-1-Nal(4)]endomorphin-2 was also the most potent analogue of the series.     

The myotropic peptides of Locusta migratoria: Structures, distribution, functions and receptors

The search for myotropic peptide molecules in the brain, corpora cardiaca, corpora allata suboesophageal ganglion complex of Locusta migratoria using a heterologous bioassay (the isolated hindgut of the cockroach, Leucophaea maderae) has been very rewarding. It has lead to the discovery of 21 novel biologically active neuropeptides. Six of the identified Locusta peptides show sequence homologies to vertebrate neuropeptides, such as gastrin/cholecystokinin and tachykinins. Some peptides, especially the ones belonging to the FXPRL amide family display pleiotropic effects. Many more myotropic peptides remain to be isolated and sequenced. Locusta migratoria has G-protein coupled receptors, which show homology to known mammalian receptors for amine and peptide neurotransmitters and/or hormones. Myotropic peptides are a diverse and widely distributed group of regulatory molecules in the animal kingdom. They are found in neuroendocrine systems of all animal groups investigated and can be recognized as important neurotransmitters and neuromodulators in the animal nervous system. Insects seem to make use of a large variety of peptides as neurotransmitters/neuromodulators in the central nervous system, in addition to the aminergic neurotransmitters. Furthermore quite a few of the myotropic peptides seem to have a function in peripheral neuromuscular synapses. the era in which insects were considered to be “lower animals” with a simple neuroendocrine system is definitely over. Neural tissues of insects contain a large number of biologically active peptides and these peptides may provide the specificity and complexity of intercellular communications in the nervous system.     

A novel and simple immunocapture assay for determination of gelatinase-b (MMP-9) activities in biological fluids: Saliva from patients with Sjögren's Syndrome contain increased latent and active gelatinase-b levels

Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys↓IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLueGly↓IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can measured directly using a chromogenic peptide substrate for urokinase. The assay has been specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this body MMp-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed.We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjögren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 ± 4.9 vs 12.2 ± 2.5 × 10⁴ cpm/ml (p > 0.05, and 44.0 (4.0 vs 36.1 ± 1.9 × 10⁴ cmp/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 ± 2.5 U/mg, controls 1.0 ± 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 ± 9.8 U/mg, controls 16.5 ± 2.6 U/mg (p = 0.01).     

Infectious Cellular Load in Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and Susceptibility of Peripheral Blood Mononuclear Cells from Their Exposed Partners to Non-Syncytium-Inducing HIV-1 as Major Determinants for HIV-1 Transmission in Homosexual Couples

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4⁺ T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 Δ32). Three of these individuals (all nonrecipients) had a CCR5 Δ32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 Δ32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.     

In vivo synthesis and processing of cereal lectins

The synthesis and processing of cereal lectins was followed in vivo. The initial translation products of lectin genes are higher molecular weight (28 K) precursors, which are post-translationally processed in a single step into authentic lectin polypeptides (23 K). The conversion of precursor into mature product is a rather slow process (the precursor has a half life of 36 min) and is apparently not a prerequisite for biological activity since the precursor exhibits sugar binding activity. Because of the striking resemblances between the processing of cereal lectins and vectorial processing of cytoplasmatically made chloroplast, mitochondrial and glyoxysomal proteins, vectorial processing of cereal lectins might be a means of transporting these proteins through a membrane into an extra-cytoplasmic compartment.