On the Significance of Cytokinin Incorporation into RNA

The clarification of the following 2 questions was attempted: (a) are cytokinins precursors in the formation of sRNA, (b) is the observed incorporation of cytokinins into sRNA related to the action of the hormone? Although Escherichia coli contains cytokinins in its sRNA, no cytokinin auxotroph mutants of E. coli could be found and the statistical probability for the existence of such mutants is extremely low. This suggests that cytokinins are not precursors in the synthesis of sRNA. A radioactive cytokinin, 6-benzylamino-9-methyl-purine was synthesized and it was tested whether or not it is incorporated into sRNA of soybean callus tissue. Masking the 9-position of the purine inhibited the incorporation of this cytokinin into RNA while not affecting its biological activity. This is taken as an indication that the observed incorporation of cytokinins such as benzyladenine into sRNA is not related to the action of this hormone.     

Effects of prenatal cocaine exposure in the retinal ganglion cell layer of the rat

To study the effects of prenatal cocaine-exposure on the developing retinal ganglion cell layer of the rat, female Wistar rats were administered subcutaneously (sc) cocaine hydrochloride (60 mg/kg body wt/d) or saline, or were not manipulated from gestational d 8–22. Male offspring were sacrificed at postnatal day 14 and 30. Radial semithin sections of epon-embedded flat mounts of the retinal quadrants were used to evaluate the following parameters along the centroperipheral axis:

1. Thickness of ganglion cell plus nerve fiber layer;
2. Nuclear size of ganglion cell layer neurons; and
3. Linear density (number per unit length) of ganglion cell layer neurons.

To study the effects of cocaine and age on the retinal areas (temporal/nasal, dorsal/ventral), a repeated measures analysis of variance was used for each of the parameters mentioned above. All parameters were affected by prenatal exposure to cocaine. The thickness of the ganglion cell plus nerve fiber layer was reduced in cocaine-exposed rats in comparison with the saline group. Nuclear diameters were smaller in the cocaine than in the saline and control groups. The linear density was higher in the cocaine-exposed group than in the control and saline groups. The linear decrease in the linear density from postnatal day 14–30 was higher in the cocaine-exposed rats than in the saline group; the decrease in the linear density along the centroperipheral axis found in both the control and saline groups was not significant in the cocaine-treated group. These morphometric findings strongly support the view that prenatal cocaine-exposure induces marked changes in the organization of the developing retina.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.     

Sensible heat transfer and thermal windows in Dasyprocta leporina (Mammalia,Rodentia)

This study aimed to evaluate the diurnal variation of the sensible heat transfer in red-rumped agoutis (Dasyprocta leporina) bred in captivity in a semi-arid environment. In addition, we seek to identify thermal windows by infrared thermography during the daytime period (07:00, 09:00, 11:00, 14:00, and 16:00). The body surface temperature was higher in the pinna (36.84 ± 0.11 °C), followed by the hind limbs (36.55 ± 0.11 °C). These body regions were primarily responsible for heat loss by radiation (which was 10.13 ± 1.17 W m^?2 and 11.19 ± 1.17 W m^?2, respectively), and acted like biological thermal windows. Heat transfer by convection was more intense in the body trunk and hind limbs at all times of the day. Thus, sensible heat transfer is important for maintaining homeothermy in red-rumped agouti in hot environments. In conclusion, these rodents use specialized body regions (pinna and hind limbs) for heat transfer.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Identification of Botryticidal Proteins with Similarity to NBS–LRR Proteins in Rosemary Pepper (<Emphasis Type="Italic">Lippia sidoides</Emphasis> Cham.) Flowers

Heavy agricultural losses are closely related to attacks by insect-pests and phytopathogens such as bacteria and fungi. Among them, the fungus Botrytis cinerea can cause gray mold in more than 200 different species of plants, and is considered a challenging problem for agribusiness. Fungicides are commonly used to control this pathogen because they are fast-working and easy to apply. However, the continuous use of fungicides may promote the selection of resistant fungi and can also cause profound contamination in ecosystems. Aiming to find alternative strategies to solve these problems, several studies have focused on searching for plant proteins and peptides with antifungal activities (AFPs). With this in mind, this report shows the isolation and characterization of two novels antifungal proteins from flowers of rosemary pepper (Lippia sidoides Cham.) with 10 and 15 kDa. Isolation was performed by using an Octyl-Sepharose hydrophobic column. In vitro bioassays indicated that isolated proteins were able to inhibit B. cinerea development, but were not effective against all bacteria tested. Moreover, N-termini sequences indicate that both proteins showed sequence homology with NBS–LRR R proteins with a lower molecular mass, suggesting possible protein fragmentation. Data reported here could help in the development of biotechnological products for crop protection against phytopathogenic fungi in the near future.     

Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.     

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.