The increased presence of repetitive motifs in the KDDR-plus recombinant protein,a kinesin-derived antigen from Leishmania infantum,improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis.     

Genetic variation for quantitative traits in soybean lines derived from tissue culture

Tissue culture may generate useful genetic variation for quantitative traits. The objective of this study was to analyze genetic variation for ten quantitative traits of soybean [Glycine max (L.) Merr.] among lines derived from the tissue culture of three cultivars. The three cultivars used to obtain R0 plants from tissue culture were BSR 101, Hodgson 78, and Jilin 3. A total of 63 R0-derived lines of BSR 101, eight of Hodgson 78, and 42 of Jilin 3 was planted with the untreated controls in row plots in a randomized complete-block design with three replications at two locations for each of 2 years. The traits evaluated were days to beginning bloom (R1), beginning seed (R5), beginning maturity (R7), full maturity (R8), height, lodging, seed yield, seed weight, protein content, and oil content. Significant (P < 0.05) variation was observed among lines for each of the ten quantitative traits. There was 57.1% of the BSR 101 lines, 87.5% of the Hodgson 78 lines, and 76.2% of the Jilin 3 lines that were significantly different from the controls for at least one trait. The percentages of lines that were significantly different from the control for an individual trait ranged from 2.7% for oil content to 25.7% for R7. The magnitude of the changes was relatively small. Although this genetic variation may be useful for cultivar development, greater variability at less expense would be expected with conventional artificial hybridization.Journal Paper No. J-14958 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IOWA, USA Project No. 2475.     

Plant regeneration in <Emphasis Type="Italic">Arachis stenosperma</Emphasis> Krapov. and W. C. Gregory from roots and calluses derived from leaflets of <Emphasis Type="Italic">in vitro</Emphasis> plants

Seed explants of A. stenosperma were cultured on MS medium supplemented with 6-benzylaminopurine with the aim of rescuing nonviable accessions stored in seed bank conditions. The regeneration potential of leaf explants from in vitro plants derived from embryonic axes was studied by using whole leaflets and leaflet segments. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine and naphthalene acetic acid. Indirect organogenesis was observed in response to 6-benzylaminopurine, either alone or in association with naphthalene acetic acid, in both explant types. Media supplemented with naphthalene acetic acid as the sole growth regulator induced rhizogenesis in whole leaflets and leaflet segments, with subsequent shoot production directly from the roots.     

Chlamydia trachomatis infection and human papillomavirus in women with cervical neoplasia in Pernambuco-Brazil

Chlamydia trachomatis (CT) is the most common bacterial cause of sexually transmitted disease. High-risk human papillomavirus (HR-HPV) is considered the main etiological agent for cervical neoplasia. Evidences showed that the presence of co-infection of CT and HR-HPV plays a central role in the etiology of cervical intraepithelial neoplasia (CIN) and cervical cancer. The goals of this study were: evaluate the human papillomavirus (HPV) and CT prevalence among Brazilian women with abnormal cytology and provide the effect of this association on the severity of cervical neoplasia. The population of this study was composed by 142 women with incident histological incidence of CIN grades I, II, III or cervical cancer from Recife, Northeast of Brazil. The polymerase chain reaction method on a cervical brush specimen was used to detect both agents and the automatic sequencing method was used for HPV genotyping assay. The prevalence of HPV and CT was 100 and 24.65 %, respectively. Thirteen types of HPV were detected; HPV 16, 18, 31 and 33 were the most common. The most prevalent HPV types were HPV 16 and 18. A significant association between CT positive and HPV 16 infection was found (p < 0.0106; OR = 5.31; 95 % IC 1.59–17.67). In the study population, there was diversity of HPV infections, with high-risk types being the most common. Also, the data collected suggest that CT infection may play an important role in the natural history of HPV infection.     

Multiple Functions of Human Papillomavirus Type 16 E6 Contribute to the Immortalization of Mammary Epithelial Cells

The E6 proteins from cervical cancer-associated human papillomavirus (HPV) types such as HPV type 16 (HPV-16) induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. We have previously shown that human mammary epithelial cells (MECs) immortalized by HPV-16 E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). To explore the potential functional significance of these interactions, we created and analyzed a series of E6 mutants for their ability to interact with E6-AP, p53, and E6BP in vitro. While there was a similar pattern of binding among these E6 targets, a subset of mutants differentiated E6-AP binding, p53 binding, and p53 degradation activities. These results demonstrated that E6 binding to E6-AP is not sufficient for binding to p53 and that E6 binding to p53 is not sufficient for inducing p53 degradation. The in vivo activity of these HPV-16 E6 mutants was tested in MECs. In agreement with the in vitro results, most of these p53 degradation-defective E6 mutants were unable to reduce the p53 level in early-passage MECs. Interestingly, several mutants that showed severely reduced ability for interacting with E6-AP, p53, and E6BP in vitro efficiently immortalized MECs. These immortalized cells exhibited low p53 levels at late passage. Furthermore, mutants defective for p53 degradation but able to immortalize MECs were also identified, and the immortal cells retained normal levels of p53 protein. These results imply that multiple functions of HPV-16 E6 contribute to MEC immortalization.     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.     

The role of the daily feeding rhythm in the regulation of the day/night rhythm in triglyceride secretion in rats

Plasma triglyceride (TG) levels show a clear daily rhythm, however, thus far it is still unknown whether this rhythm results from a daily rhythm in TG production, TG uptake or both. Previous studies have shown that feeding activity affects plasma TG concentrations, but it is not clear how the daily rhythm in feeding activity affects plasma TG concentrations. In the present study, we measured plasma TG concentrations and TG secretion rates in rats at 6 Zeitgeber times to investigate whether plasma TG concentrations and TG secretion show a daily rhythm. We found that plasma TG concentrations and TG secretion show a significant day/night rhythm. Next, we removed the daily rhythm in feeding behavior by introducing a 6-meals-a-day (6M) feeding schedule to investigate whether the daily rhythm in feeding behavior is necessary to maintain the daily rhythm in TG secretion. We found that the day/night rhythm in TG secretion was abolished under 6M feeding conditions. Hepatic apolipoprotein B (ApoB) and microsomal TG transfer protein (Mttp), which are both involved in TG secretion, also lost their daily rhythmicity under 6M feeding conditions. Together, these results indicate that: (1) the daily rhythm in TG secretion contributes to the formation of a day/night rhythm in plasma TG levels and (2) a daily feeding rhythm is essential for maintaining the daily rhythm in TG secretion.