Conformational changes of L-rRNA during elongation of polypeptide

A ribosome undergoes significant conformational changes during elongation of a polypeptide chain, and these are correlated with structural changes of rRNAs. We tested 15 different oligonucleotides complementary to the selected, highly conserved seqences of rRNAs (L-rRNA, 5S rRNA and tRNA) important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site and a polymerization of polypeptide chains on the ribosomes converted either to pre- or to posttranslocational states. The inhibition of polymerization reaction by complementary oligonucleotides was high in all ribosomal states. The efficiency of inhibition of binding reaction was lower and more diverse than was the polypeptide elongation. We conclude that the selected oligonucleotides inhibit polypeptide synthesis with different effectivity, primarily depending on L-rRNA conformation within ribosome architecture.     

Presence of maternal anti-HBs antibodies does not influence hepatitis B vaccine response in Brazilian neonates

Recently, it was suggested that maternal hepatitis B surface antigen antibodies (anti-HBs) acquired transplacentally could play a negative role in newborn infants' immune response to the hepatitis B vaccine. We compared the hepatitis B virus (HBV) vaccine response in infants born to mothers previously vaccinated against HBV (n = 91) to infants born to mothers who were not previously vaccinated (n = 221). All newborn infants received three intramuscular doses (10 μg) of HBV vaccine (Butang?) at 0,1 and six months. The first dose was administered at the maternity hospital within 12 h of birth. The geometric mean titres of anti-HBs were not different among newborn infants born to mothers who were anti-HBs-negative (492.7 mIU/mL) and anti-HBs-positive (578.7 mIU/mL) (p = 0.38). Eight infants did not respond to the HBV vaccine. Of them, six were born to anti-HBs-negative mothers and two were born to mothers with anti-HBs titres less than 50 mlU/mL. Despite the mother's anti-HBs-positive status, our data show a good immunogenicity of the Brazilian HBV recombinant vaccine in neonates.     

Improved radiosynthesis and preliminary in vivo evaluation of the 11C-labeled tetrazine [11C]AE-1 for pretargeted PET imaging

Pretargeted nuclear imaging based on the ligation between tetrazines and nano-sized targeting agents functionalized with trans-cyclooctene (TCO) has recently been shown to improve both imaging contrast and dosimetry in nuclear imaging of nanomedicines. Herein, we describe the improved radiosynthesis of a ¹¹C-labeled tetrazine ([¹¹C]AE-1) and its preliminary evaluation in both mice and pigs. Pretargeted imaging in mice was carried out using both a new TCO-functionalized polyglutamic acid and a previously reported TCO-functionalized bisphosphonate system as targeting agents. Unfortunately, pretargeted imaging was not successful using these targeting agents in pair with [¹¹C]AE-1. However, brain imaging in pig indicated that the tracer crossed the blood-brain-barrier. Hence, we suggest that this tetrazine scaffold could be used as a starting point for the development of pretargeted brain imaging, which has so far been a challenging task.     

Infection with intestinal helminth (Hymenolepis diminuta) impacts exploratory behavior and cognitive processes in rats by changing the central level of neurotransmitters

Parasites may significantly affect the functioning of the host organism including immune response and gut-brain-axis ultimately leading to alteration of the host behavior. The impact of intestinal worms on the host central nervous system (CNS) remains unexplored. The aim of this study was to evaluate the effect of intestinal infection by the tapeworm Hymenolepis diminuta on behavior and functions of the CNS in rats. The 3 months old animals were infected, and the effects on anxiety, exploration, sensorimotor skills and learning processes were assessed at 18 months in Open Field (OF), Novel Object Recognition (NOR) and the Water Maze (WM) tests. After completing the behavioral studies, both infected and non-infected rats were sacrificed, and the collected tissues were subjected to biochemical analysis. The levels of neurotransmitters, their metabolites and amino acids in selected structures of the CNS were determined by HPLC. In addition, the gene expression profile of the pro- and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) was evaluated by Real-Time PCR to determine the immune response within the CNS to the tapeworm infection. The parasites caused significant changes in exploratory behavior, most notably, a reduction of velocity and total distance moved in the OF test; the infected rats exhibited decreased frequency in the central zone, which may indicate a higher level of anxiety. Additionally, parasite infestation improved spatial memory, assessed in the WM test, and recognition of new objects. These changes are related to the identified reduction in noradrenaline level in the CNS structures and less pronounced changes in striatal serotonergic neurotransmission. H. diminuta infestation was also found to cause a significant reduction of hippocampal expression of IL-6. Our results provide new data for further research on brain function during parasitic infections especially in relation to helminths and diseases in which noradrenergic system may play an important role.     

Inbreeding by environmental interactions affect gene expression in Drosophila melanogaster

Genomewide gene expression patterns were investigated in inbred and noninbred Drosophila melanogaster lines under benign and stressful (high temperature) environmental conditions in a highly replicated experiment using Affymetrix gene chips. We found that both heat-shock protein and metabolism genes are strongly affected by temperature stress and that genes involved in metabolism are differentially expressed in inbred compared with noninbred lines, and that this effect is accentuated after heat stress exposure. Furthermore we show that inbreeding and temperature stress cause increased between-line variance in gene expression patterns. We conclude that inbreeding and environmental stress both independently and synergistically affect gene expression patterns. Interactions between inbreeding and the environment are often observed at the phenotypic level and our results reveal some of the genes that are involved at the individual gene level. Our observation of several metabolism genes being differentially expressed in inbred lines and more so after exposure to temperature stress, together with lower fitness in the investigated inbred lines, supports the hypothesis that superiority of heterozygous individuals partly derives from increased metabolic efficiency.     

Characterization of Primary and Secondary Cultures of Astrocytes Prepared from Mouse Cerebral Cortex

Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary acidic protein, and the glutamate transporters EAAT1 and EAAT2. The cultures prepared from 7-day-old animals were additionally characterized metabolically using (13)C-labeled glucose and glutamate as well as (15)N-labeled glutamate as substrates. All types of cultures exhibited pronounced immunostaining of the astrocyte marker proteins. The metabolic pattern of the cultures from 7-day-old animals of the labeled substrates was comparable to that seen previously in astrocyte cultures prepared from new-born mouse brain showing pronounced glycolytic and oxidative metabolism of glucose. Glutamate was metabolized both via the GS pathway and oxidatively via the tricarboxylic acid cycle as expected. Additionally, glutamate underwent pronounced transamination to aspartate and alanine and the intracellular pools of alanine and pyruvate exhibited compartmentation. Altogether the results show that cultures prepared from cerebral cortex of 7-day-old mice have metabolic and functional properties indistinguishable from those of classical astrocyte cultures prepared from neocortex of new-born animals. This provides flexibility with regard to preparation and use of these cultures for a variety of purposes.     

Structural and functional insight into how the Plasmodium falciparum VAR2CSA protein mediates binding to chondroitin sulfate A in placental malaria

Malaria is a major global health problem. Pregnant women are susceptible to infection regardless of previously acquired immunity. Placental malaria is caused by parasites capable of sequestering in the placenta. This is mediated by VAR2CSA, a parasite antigen that interacts with chondroitin sulfate A (CSA). One vaccine strategy is to block this interaction with VAR2CSA-specific antibodies. It is a priority to define a small VAR2CSA fragment that can be used in an adhesion blocking vaccine. In this, the obvious approach is to define regions of VAR2CSA involved in receptor binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with nanomolar affinity, and that the CSA-binding site lies in the N-terminal part of the protein. In this study we define the minimal binding region by truncating VAR2CSA and analyzing CSA binding using biosensor technology. We show that the core CSA-binding site lies within the DBL2X domain and parts of the flanking interdomain regions. This is in contrast to the idea that single domains do not possess the structural requirements for specific CSA binding. Small-angle x-ray scattering measurements enabled modeling of VAR2CSA and showed that the CSA-binding DBL2X domain is situated in the center of the structure. Mutating classic sulfate-binding sites in VAR2CSA, along with testing dependence of ionic interactions, suggest that the CSA binding is not solely dependent on the sulfated CSA structure. Based on these novel PfEMP1 structure-function studies, we have constructed a small VAR2CSA antigen that has the capacity to induce highly adhesion-blocking antibodies.     

Human cells: new platform for recombinant therapeutic protein production

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production.