Differential kinetics and inhibition of purified recombinant tyrosine kinase 2 (TYK-2) and its catalytic domain JH-1

The Janus kinase (JAK) family consists of four members: JAK-1, -2, -3 and tyrosine kinase 2 (TYK-2). Recent work suggests that cytokine signaling through TYK-2 may play a critical role in a number of inflammatory processes. We recently described the purification and characterization of phosphorylated isoforms of the TYK-2 kinase domain (TYK-2 KD) and its high resolution 3D structure in the presence of inhibitors. We now report the expression and a two-step purification procedure for the doubly tagged full-length construct, H6-FL-TYK-2-FLAG, and examine its properties compared to those of TYK-2 KD. In the presence of ATP and a peptide substrate, H6-FL-TYK-2-FLAG showed a marked lag in phosphopeptide product formation, while TYK-2 KD showed no such lag. This lag could be eliminated by ATP pretreatment, suggesting that the H6-FL-TYK-2-FLAG enzyme was activated by phosphorylation. The potencies of several nanomolar inhibitors were similar for TYK-2 KD and H6-FL-TYK-2-FLAG. However, these same inhibitors were about 1000 times less potent inhibiting the autophosphorylation of H6-FL-TYK-2-FLAG than they were inhibiting the phosphorylation of a peptide substrate modeled after the activation loop sequence of TYK-2. This intriguing result suggests that autophosphorylation and, thus, activation of H6-FL-TYK-2-FLAG is relatively insensitive to inhibition and that present inhibitors act to inhibit TYK-2 subsequent to activation. Inhibition of TYK-2 autophosphorylation may represent a new area of investigation for the JAK family.     

Galvechelone lopezmartinezae gen. et sp. nov., a new cryptodiran turtle in the Lower Cretaceous of Europe

Abstract: The Spanish town of Galve (Teruel) is notable because of the abundance of Upper Jurassic and, especially, Lower Cretaceous vertebrates recorded there. Although most groups have been studied in detail, information on turtles is very limited even though the material is relatively abundant. So far, no turtle taxa have been identified at the generic level. The only Lower Cretaceous articulated specimen from Galve is analysed here. It is identified as a representative of Cryptodira, Galvechelone lopezmartinezae gen. et sp. nov. Galvechelone lopezmartinezae is determined as a taxon belonging to the node that groups the turtles traditionally assigned to ‘Macrobaenidae’ and ‘Sinemydidae’, and other taxa such as the members of Panchelonioidea. This node, very abundant in the Lower Cretaceous of Asia, and with a broad subsequent distribution, has recently been recognized in the Lower Cretaceous of Europe. The diversity of basal members of Eucryptodira in the European Late Jurassic (represented by Thalassemydidae, Plesiochelyidae and Eurysternidae) was high. Owing to a relative scarcity of well‐preserved early Cretaceous turtles from Europe, the knowledge of this group of reptiles is limited. The study of the new turtle from Galve, together with the recently described Hoyasemys jimenezi, and the recently completed review of the enigmatic Chitracephalus dumonii demonstrate that members of the cryptodiran node grouping ‘Macrobaenidae’, ‘Sinemydidae’ and Panchelonioidea were also very diverse in this period.     

Some physicochemical properties of hemoglobin-manitoba (alpha2 102Ser replaced by Arg (G9) beta2).

Hb-Manitoba was discovered in 1970 [1] in a Canadian family of British origin. Recently we observed the same variant in a second family, and found that the oxy-derivative of Hb-Manitoba is slightly unstable at 65 degrees C, dissociates less readily at alkaline pH than does Hb-A, and forms asymmetric hybrids with other hemoglobins which are readily detectable by electrophoresis.     

Improving ability to identify malaria and correctly use chloroquine in children at household level in Nakonde District, Northern Province of Zambia

BACKGROUND: This study investigated causes of malaria and how cases were managed at household level, in order to improve the ability to identify malaria and ensure correct use of chloroquine. It was conducted in Nakonde District, Northern Province of Zambia, between 2000 and 2001. Nakonde district is in a hyperendemic malaria province, where Plasmodium falciparum is predominant. The district has a total population of 153, 548 people, the majority of whom are peasant farmers. The main aim of the post intervention survey was to establish the proportion of caretakers of children five years and below, who were able to identify simple and severe malaria and treat it correctly using chloroquine in the home. METHODS: A baseline survey was conducted in five wards divided into intervention and control.Intervention and control wards were compared. Village health motivators and vendors were identified and trained in three intervention wards, as a channel through which information on correct chloroquine dose could be transmitted. A total of 575 carers, who were 15 years old and above and had a child who had suffered from malaria 14 days before the survey commenced, were interviewed. The two control wards received no intervention. 345 caretakers were from the intervention wards, while 230 came from the control wards. Identification of malaria and correct use of anti-malarial drugs was assessed in terms of household diagnosis of malaria in children under five years, type and dose of anti-malarial drugs used, self medication and the source of these anti-malarials. RESULTS: The majority of respondents in the study were females (81%). Chloroquine was the most frequently used anti-malarial (48.5%) in both the intervention and control wards. There was no difference between the intervention and control wards at pre-intervention (P = 0.266 and P = 0.956), in the way mothers and other caretakers identified simple and severe malaria. At baseline, knowledge on correct chloroquine dosage in the under five children was comparable between intervention and control wards. Post-intervention revealed that mothers and other caretakers were 32% and 51%, respectively, more likely to identify simple and severe malaria. There was a 60% increase on correct chloroquine dosage in all age groups among carers living in post-intervention wards. CONCLUSION: Compliance with standard therapeutic doses and correct identification of malaria was poorest in control wards, where no motivators and vendors were trained.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Trichoderma spp. and Bacillus spp. as growth promoters in maize (Zea mays L.)

Microbes that are beneficial to plants are used to enhance the crop growth, yield and are alternatives to chemical fertilizers. Trichoderma and Bacillus are the predominant plant growth-promoting fungi and bacteria. The objective of this study was select, characterize, and evaluate isolates of Trichoderma spp. and Bacillus spp. native from the northern region of Sinaloa, Mexico, and assess their effect on growth promotion in maize (Zea mays L.). In greenhouse conditions, four Trichoderma isolates and twenty Bacillus isolates, as well as two controls, were tested in a completely randomized design with three replicates. We selected the two best strains of Trichoderma and Bacillus: TB = Trichoderma asperellum, TF = Trichoderma virens, B14 = Bacillus cereus sensu lato and B17 = Bacillus cereus, which were evaluated in the field in a completely randomized blocks in factorial arrangement design with three replicates applying different rates of nitrogen fertilizer (0, 150 kg N/ha, and 300 kg N/ha). Treatments 5 (B17 = B. cereus) and 11 (TF = T. virens) both fertilized with 150 kg N/ha showed similar yields and they did not reveal significant differences from the treatments fertilized with 300 kg N/ha. This indicated that treatment 5 (B17= B. cereus with 150 kg N/ha) and treatment 11 (TF= T. virens with 150 kg N/ha) were efficient as growth promoters, by not showing significant differences in root volume and dry weight of foliage. The results indicated a reduction of 50% in the rate of nitrogen to fertilizer required for maize (Zea mays L.) crops. These microorganisms Trichoderma and Bacillus could be an alternative to reduce the use of chemical fertilizers in maize.