Toxoplasma gondii regulates ICAM-1 mediated monocyte adhesion to trophoblasts

Materno-foetal transmission causes one of the most serious forms of infection with the intracellular protozoan parasite Toxoplasma gondii. In the placenta, trophoblast cells constitute the barrier between maternal circulation and foetal tissue. We looked at the factors that determine the extent of cell adhesion to human BeWo trophoblast cells during T. gondii infection. BeWo monolayers stimulated with the supernatant of T. gondii-infected PBMC showed a large increase in THP-1 cell adhesion and upregulation of the intercellular adhesion molecule (ICAM)-1. Neutralization of cytokines by corresponding antibodies demonstrated that anti-IFN-gamma, but not anti-TNF-alpha or anti-IL-1beta, led to a significant reduction of THP-1 adhesion to a BeWo monolayer. Treatment of BeWo cells with single cytokines failed to induce upregulation of adhesion. In contrast, simultaneous treatment with IFN-gamma and either TNF-alpha or IL-1beta mimicked strongly the effect of infected cell supernatant. The results suggest that IFN-gamma plays a pivotal role in the cell adhesion process through upregulation of ICAM-1 and in the process of congenital transmission of T. gondii.     

Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines

'High-risk' human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumours therapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, we selected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalently on pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16 transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel of normal or transformed cell lines. Two of the three selected peptides targeted cells only when presented on phage particles in a constrained conformation. However, all three peptides retained their targeting capacity when presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form. These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervical cancer.     

Patterns of male scent-marking in Propithecus edwardsi of Ranomafana National Park, Madagascar

Scent-marking behavior has been well documented in many primate species. Three common functions attributed to scent-marking in males of multi-male/multi-female lemur species include: 1) advertisement of individual identity, 2) territorial defense, and 3) reproductive suppression. We examined the average number of scent-marks per hour exhibited daily by adult male sifakas (Propithecus edwardsi) and found that patterns of scent-marking changed with season, natal status, and dominance status. Males in single-male groups scent-marked at the highest rate, followed by dominant males, males of equal status, and subordinate males. Non-natal males generally scent-marked at higher rates than natal males, and adult males living in a natal group without a parent marked at higher rates than males living with a parent. All males scent-marked at higher rates in the migration season compared to the other seasons. These patterns were consistent with territorial defense and advertisement to females, and the suggestion that these chemical signals impart information concerning status. Since scent-marking behavior tracked seasons and varied with both dominance and natal status, it may serve multiple functions in males.     

Development of a large-scale chemogenomics database to improve drug candidate selection and to understand mechanisms of chemical toxicity and action

Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.     

Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1

Background

Plasmodium vivax is the second most prevalent malaria parasite affecting more than 75 million people each year, mostly in South America and Asia. In addition to major morbidity this parasite is associated with relapses and a reduction in birthweight. The emergence and spread of drug resistance in Plasmodium falciparum is a major factor in the resurgence of this parasite. P. vivax resistance to drugs has more recently emerged and monitoring the situation would be helped, as for P. falciparum, by molecular methods that can be used to characterize parasites in field studies and drug efficacy trials.

Methods

Practical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers, Pvcs and Pvmsp1, were developed. The methodology was evaluated using 100 P. vivax isolates collected in Thailand.

Results and Discussion

Analysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29). A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1. These were generally randomly distributed amongst the isolates. A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1.

Conclusion

These results indicate that the genotyping protocols presented can be useful in the assessment of in vivo drug efficacy clinical trials conducted in endemic areas and for epidemiological studies of P. vivax infections.     

Prenatal cocaine alone and combined with nicotine alters ANG II and IGF-1 induced left atrial contractions in aging male offspring

Prenatal cocaine or nicotine affects inotropic activity in the hearts of rat offspring. However, the long-term consequence of this exposure on the cardiac response to hormonal challenge is unknown. We assessed the inotropic effects of angiotensin II (ANG II) and insulin-like growth factor 1 (IGF-1) in the left atria of 19.0-24.5 month-old male rats exposed on gestation days 8-21 to 1 of 6 treatments: low cocaine (LC) (20 mg/kg) or high cocaine (HC) (40 mg/kg); 20 mg/kg cocaine and high nicotine (5 mg/kg nicotine) (LC/HN); 40 mg/kg cocaine and low nicotine (2.5 mg/kg nicotine) (HC/LN); pair fed: yoked to HC (PF); saline: injection of 0.9% NaCl (SAL). Isometric contractions were assessed by electrical stimulation of isolated left atria superfused with Tyrode solution (control) to which ANG II (10-7 mol/L, 20 min) and IGF-1 (10-8 mol/L, 20 min) in the presence of ANG II were added sequentially. Offspring in all cocaine groups showed a higher peak tension development (PTD) to ANG II than PF controls. This increase in PTD was attenuated by subsequent addition of IGF-1 in all except HC offspring. However, with the HC/LN combination the IGF-1 effect on PTD was again evident. The velocities of contraction and relaxation were positively affected by ANG II only in the combined prenatal drug groups; IGF-1 reduced only contraction velocity. Our data demonstrate that IGF-1 reverses the positive inotropic effect of ANG-II in atrial muscle of aging rats and that gestational exposure to only high doses of cocaine eliminates this protective response. It appears that combined prenatal exposure to cocaine and nicotine does not exacerbate the decline in cardiac function and responsiveness to inotropic drugs seen in the aging heart.     

ANT2 Isoform Required for Cancer Cell Glycolysis

The three adenine nucleotide translocator ({ANT1} to {ANT3}) isoforms, differentially expressed in human cells, play a crucial role in cell bioenergetics by catalyzing ADP and ATP exchange across the mitochondrial inner membrane. In contrast to differentiated tissue cells, transformed cells, and their ρ⁰ derivatives, i.e. cells deprived of mitochondrial DNA, sustain a high rate of glycolysis. We compared the expression pattern of {ANT} isoforms in several transformed human cell lines at different stages of the cell cycle. The level of {ANT2} expression and glycolytic ATP production in these cell lines were in keeping with their metabolic background and their state of differentiation. The sensitivity of the mitochondrial inner membrane potential (Δψ) to several inhibitors of glycolysis and oxidative phosphorylation confirmed this relationship. We propose a new model for ATP uptake in cancer cells implicating the {ANT2} isoform, in conjunction with hexokinase II and the β subunit of mitochondrial ATP synthase, in the Δψ maintenance and in the aggressiveness of cancer cells.     

AIP4 restricts transforming growth factor-beta signaling through a ubiquitination-independent mechanism

Smad7 functions as an intracellular antagonist in transforming growth factor-beta (TGF-beta) signaling. In addition to interacting stably with the activated TGF-beta type I receptor (TbetaRI) to prevent phosphorylation of the receptor-regulated Smads (Smad2 and Smad3), Smad7 also induces degradation of the activated TbetaRI through association with different E3 ubiquitin ligases. Using the two-hybrid screen, we identified atrophin 1-interacting protein 4 (AIP4) as an E3 ubiquitin ligase that specifically targets Smad7 for ubiquitin-dependent degradation without affecting the turnover of the activated TbetaRI. Surprisingly, we found that despite the ability to degrade Smad7, AIP4 can inhibit TGF-beta signaling, presumably by enhancing the association of Smad7 with the activated TbetaRI. Consistent with this notion, expression of a catalytic mutant of AIP4, which is unable to induce ubiquitination and degradation of Smad7, also stabilizes the TbetaRI.Smad7 complex, resulting in inhibition of TGF-beta signaling. The ability of AIP4 to enhance the inhibitory function of Smad7 independent of its ubiquitin ligase activity reveals a new mechanism by which E3 ubiquitin ligases may function to turn off TGF-beta signaling.     

Protein structural perturbation and aggregation on homogeneous surfaces

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by alpha-helix to turns/random during the first 30 min and followed by a slow alpha-helix to beta-sheet transition. Increase in intramolecular and intermolecular beta-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with -CH3, -OPh, -CF3, -CN, -OCH3, and -OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces.