Characterization of RAB-like4, the first identified RAB-like protein from Trypanosoma cruzi with GTPase activity

RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.     

Transmission of “Candidatus Phytoplasma pruni”‐related strain associated with broccoli stunt by four species of leafhoppers

A disease known as broccoli stunt, associated with “Candidatus Phytoplasma pruni”‐related strain, has been responsible by significant economic losses in crops grown in the State of São Paulo, Brazil. Previous investigations evidenced some species of leafhoppers observed in broccoli fields as potential vectors of the phytoplasma. In this study, the six species more frequently found in broccoli crops were collected to confirm that evidence. Group of five insects of each species were confined per broccoli seedling for an inoculation access period (IAP) of 48 hr. After the IAP, each group was tested for detection of phytoplasma. Evaluation of plants was performed 60 days after inoculation based on the presence of phytoplasma in their tissues. When transmission was positive, genomic fragments corresponding to 16S rDNA were sequenced both for the infected plants and its respective group of insects. The results revealed that the species Agallia albidula, Agalliana sticticollis, Atanus nitidus and Balcluta hebe were able to transmit phytoplasma to broccoli seedlings. Based on the estimates of transmission probability by single insects (P), the highest transmission rate was observed for A. nitidus (24.2%) and the lowest for B. hebe (1.9%). The sequencing of 16S rDNA revealed complete similarity between the sequences of the phytoplasma transmitted to broccoli test plants and the sequences of the phytoplasma found in the field‐collected leafhoppers. These findings support the inclusion of those species as vectors of phytoplasmas belonging to 16SrIII group in broccolis, providing additional information to improve management of this important disease of endemic occurrence.     

Protein biosynthesis changes in Trypanosoma cruzi induced by supra-optimal temperature

Early during vertebrate infection, T. cruzi is exposed to the host blood at an elevated temperature. Bearing this in mind, the pattern of protein synthesis of two parasite forms was examined. SDS-PAGE of heated organisms showed an increase in at least four proteins (103, 92, 75 and 61 kD). The temperature effect is also manifested in cells whose RNA synthesis is reduced by actinomycin D treatment. The synthesis of the '29 degrees proteins' is inhibited at 40 degrees C in organisms growing in culture medium; when the organisms were maintained in serum, the inhibition was not observed. The inhibitory effect observed at 40 degrees C was reversed when the temperature was shifted to 29 degrees C. These proteins were synthesized for 180 min at 37 degrees C or 360 min at 40 degrees C. The increased protein synthesis manifested at 37 degrees C had decreased 45 min after the temperature was lowered to 29 degrees C. When the cells were pre-incubated at 40 degrees C and shifted to 29 degrees C, the synthesis of the heat-induced proteins proceeded for at least 180 min. This pattern of heat induction in epimastigotes and trypomastigotes is the same irrespective of whether the incubation medium is LIT (for epimastigotes), M-16 (for trypomastigotes), or when serum was used for both cell types.     

Poly(A) RNA metabolism during change of physiological state of Tetrahymena pyriformis cells

In the present work the metabolism of poly(A)+ RNA was investigated in cells of Tetrahymena pyriformis derived either from stationary cultures or from starved suspensions that were initiating growth. Under these circumstances the organisms derived from stationary cultures synthesize ribosomal and poly(A)+ RNA and form polysomes. In the presence of actinomycin D (actD) the observed expansion of the polysomal population is arrested. Pre-starved cells, on the other hand, start making polysomes in the virtual absence of ribosomal and poly(A)+ RNA synthesis soon after being transferred to peptone medium. In this case polysome formation is only partially sensitive to actD. These results have been interpreted as indicating that, in the beginning of growth, cells derived from stationary cultures are dependent on RNA synthesis for polysome formation, whereas pre-starved cells use pre-synthesized RNA for the same purpose.     

Gold Nanohole Arrays Fabricated by Interference Lithography Technique as SERS Probes for Chemical Species Such As Rhodamine 6G and 4,4′-Bipyridine

In the present study, we report the SERS effect of rhodamine 6G (Rh6G) and 4-4′ bipyridine (Bipy) molecules on the surface of gold nanohole (AuNh) arrays fabricated by the interference lithography (IL) technique. It was observed an enhancement of the main bands of Rh6G and 4-4′ Bipy molecules, when deposited over the surface of AuNh, in comparison when that was deposited on the surface of 5-nm flat gold film. Our study indicates that metallic nanostructures fabricated by IL technique have the potential for high impact on biochemical sensing, such as DNA and bacterial detection, real time glucose sensing for diabetes, and in situ identification of reaction products.     

H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing

DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.