Lead toxicity in Saccharomyces cerevisiae

The effect of Pb on Saccharomyces cerevisiae cell structure and function was examined. Membrane integrity was assessed by the release of UV-absorbing compounds and by the intracellular K⁺ efflux. No leakage of UV₂₆₀-absorbing compounds or loss of K⁺ were observed in Pb (until 1,000 μmol/l) treated cells up to 30 min; these results suggest that plasma membrane seems not to be the immediate and primary target of Pb toxicity. The effect of Pb on yeast metabolism was examined using the fluorescent probe FUN-1 and compared with the ability to reproduce, evaluated by colony-forming units counting. The exposition of yeast cells, during 60 min to 1,000 μmol/l Pb, induces a decrease in the ability to process FUN-1 although the cells retain its proliferation capacity. A more prolonged contact time (120 min) of yeast cells with Pb induces a marked (> 50%) loss of yeast cells metabolic activity and replication competence through a mechanism which most likely requires protein synthesis.     

Expression of Toll-like receptors (TLR) and responsiveness to TLR agonists by polarized mouse uterine epithelial cells in culture

The objective of the present study was to examine the expression of Toll-like receptors (TLRs) by mouse uterine epithelial cells and to determine if stimulation of the expressed TLR induces changes in cytokine and/or chemokine secretion. Using RT-PCR, the expression of TLRs 1-6 by mouse uterine epithelial cells was demonstrated, with TLRs 7-9 expressed only periodically. In the absence of pathogen-associated molecular patterns, polarized uterine epithelial cells constitutively secrete interleukin (IL) 1A, cysteine-cysteine ligand (CCL) 2, IL6, granulocyte-macrophage colony-stimulating factor 2 (CSF2), tumor necrosis factor A (TNFA), CSF3, and IL8 in vitro, with levels of cytokines/chemokines secreted into the apical compartment being significantly greater than those released into the basolateral compartment. When added to the apical surface for 48 h before analysis, the TLR2-agonist Pam3Cys-Ser-(Lys)4 and TLR1/6-agonist peptidoglycan increased epithelial cell apical secretion of IL1A, CCL2, and IL6 and apical/basolateral bidirectional secretion of CSF2, TNFA, CSF3, and IL8 when compared to controls. The TLR3-agonist poly (I:C) significantly increased bidirectional secretion of CCL2, IL6, TNFA, and CSF2 and basolateral secretion of CSF3. Lastly, the TLR4-agonist lipopolysaccharide increased bidirectional secretion CCL2, CSF2, TNFA, CSF3, and IL8 and apical secretion of IL6. These results indicate that mRNAs for Tlr1 through Tlr6 are expressed by uterine epithelial cells and that treatment with specific TLR agonists alters the expression of key chemokines and proinflammatory cytokines that contribute to the defense of the uterus against potential pathogens.     

Social structures and social relations--an archaeological and anthropological examination of three early Medieval separate burial sites in Bavaria

As part of an interdisciplinary cooperation, three early medieval separated burial sites from central Bavaria were subjected to archaeological and anthropological examinations. Separated burial sites are probably an expression of social stratification. This paper focuses on two characteristic aspects of these separated burial sites: (1) structure of the site, and (2) biological manifestation of a social upper class. The separated burial grounds Etting/Sandfeld (31 individuals), Grossmehring (44 individuals) and Kelheim (43 individuals), all located in southern Bavaria, were analyzed. Sex and age at death of all individuals were determined, and dietary behaviour was reconstructed by means of carbon and nitrogen isotope ratios in bone collagen. Local conditions such as climate, soil conditions or the intensity of agriculture can provoke significant variations in plant delta15N-values, which could lead to a shift of the baseline values of the corresponding trophic web. To facilitate the comparison of isotopic data from different sites, delta15N values of cattle bones were taken as a reference for the human data (presuming the diet to consumer chain). The results of dietary reconstruction indicate that the populations enjoyed very good living conditions with a primarily animal protein-based diet. Furthermore, the isotope analysis revealed more detailed indications for certain individuals regarding their social status. Some individuals can even be appointed as possible chiefs of the population, since anthropological and archaeological interpretations were in total agreement: It was possible to identify persons of a higher social status based on the structure of the burial site, grave goods and the isotopic analysis.     

Evidence for novel loci for late-onset Parkinson’s disease in a genetic isolate from the Netherlands

We studied patients with idiopathic Parkinson’s disease (PD) from an isolated population in the Netherlands aiming to map gene(s) involved in PD susceptibility. A total of 109 parkinsonism patients were independently ascertained, of whom 62 presented late-onset, idiopathic PD. Genealogical research showed that 45 index cases with idiopathic PD were linked to a common ancestor, indicating familiar clustering among the patients. This strong familial clustering was highly significant (P=0.005) when compared to random controls from the same population. We performed a genome wide scan using 382 polymorphic markers in 44 distantly related PD patients plus 112 unaffected first-degree relatives and spouses. Our genome wide association analysis (DISLAMB) revealed evidence of association at a nominal P-value<0.01 for markers D2S2333, D4S405, D9S158, D13S153. Other regions on chromosomes 3p, 4q, 14q, 17p and 17q were found at a significance level of P<0.05. In a follow-up study, we investigated all the positive regions using a denser marker set and a larger sample (total of 630 individuals including all late-onset PD patients). The strongest evidence for association remained for the 9q and 14q region. A significant association was found for marker D9S1838 (OR=2.0, 95% CI 1.1–3.5, P=0.014) and D14S65 (OR=3.2, 95% CI 1.7–6.1, P<0.001). Moreover, a common haplotype with excess of sharing among late-onset PD cases was observed on both regions. Our results suggest the existence of two loci influencing PD susceptibility on chromosome 9q and 14q.     

An antibody-based microarray assay for small RNA detection

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 µl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.     

Contribution of mesenterial muscle dedifferentiation to intestine regeneration in the sea cucumber Holothuria glaberrima

Holothurians (sea cucumbers) have been known from ancient times to have the capacity to regenerate their internal organs. In the species Holothuria glaberrima, intestinal regeneration involves the formation of thickenings along the free mesentery edge; these thickenings will later give rise to the regenerated organ. We have previously documented that a remodeling of the extracellular matrix and changes in the muscle layer occur during the formation of the intestinal primordium. In order to analyze these changes in depth, we have now used immunocytochemical techniques and transmission electron microscopy. Our results show a striking disorganization of the muscle layer together with myocyte dedifferentiation. This dedifferentiation involves nucleic activation, disruptions of intercellular junctions, and the disappearance of cell projections, but more prominently, the loss of the contractile apparatus by the formation and elimination of spindle-like structures. Muscle dedifferentiation can be seen as early as 2 days following evisceration and continues during the next 2 weeks of the regeneration process. Dedifferentiation of myocytes might result in cells that proliferate and give rise to new myocytes. Alternatively, dedifferentiating myocytes could give rise to cells with high nuclear-to-cytoplasmic ratios, with some being eliminated by apoptosis. Our results, together with those in other regeneration models, show that myocyte dedifferentiation is a common event in regeneration processes and that the dedifferentiated cells might play an important role in the formation of the new tissues or organs. This work was supported by NSF (IBN-0110692) and NIH-MBRS (S06GM08102). We also acknowledge partial support from RCMI (RRO-3641-01), the Department of Biology, and the University of Puerto Rico.     

Fast-disintegrating sublingual tablets: Effect of epinephrine load on tablet characteristics

The aim of this study was to evaluate the effect of increasing epinephrine load on the characteristics of fast-disintegrating sublingual tablets for the potential emergency treatment of anaphylaxis. Four tablet formulations, A, B, C, and D, containing 0%, 6%, 12%, and 24% of epinephrine bitartrate, respectively, and microcrystalline cellulose:low-substituted hydroxypropyl cellulose (9∶1), were prepared by direct compression, at a range of compression forces. Tablet weight variation, content uniformity, hardness, disintegration time, wetting time, and friability were measured for each formulation at each compression force. All 4 tablet formulations at each compression force were within the United States Pharmacopeia (USP) limits for weight variation and content uniformity. A linear increase in compression force resulted in an exponential increase in hardness for all formulations, a linear increase in disintegration and wetting times of A, and an exponential increase in disintegration and wetting times of B, C, and D. At a mean±SD hardness of ≥2.3±0.2 kg, all tablet formulations passed the USP friability test. At a mean±SD hardness of ≤3.1±0.2 kg, all tablet formulations resulted in disintegration and wetting times of <10 seconds and <30 seconds, respectively. Tablets with drug loads from 0% to 24% epinephrine can be formulated with hardness, disintegration times, and wetting times suitable for sublingual administration.     

Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice

Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, approximately threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, metacyclic forms of isolate 573, like the CL isolate, produced high parasitemia (>10(6) parasites per ml at the peak), killing approximately 40% of animals, whereas infection with isolate 587 resulted in low parasitemia (<10(5) parasites per ml), with zero mortality. On the fourth day post-inoculation, tissue sections of the mouse stomach stained with hematoxylin and eosin showed a four to sixfold higher number of epithelial cells infected with isolate 573 or CL than with isolate 587. The rate of intracellular parasite development was similar in all isolates. Mimicking in vivo infection, parasites were treated with pepsin at acidic pH and then assayed for their ability to enter HeLa cells or explanted gastric epithelial cells. Pepsin extensively digested gp90 from isolate 573 and significantly increased invasion of both cells, but had minor effect on gp90 or infectivity of isolates 587 and CL. The profile of g82 digestion was similar in isolates 573 and 587, with partial degradation to a approximately 70 kDa fragment, which preserved the target cell binding domain as well as the region involved in gastric mucin adhesion. Gp82 from CL isolate was resistant to pepsin. Assays with parasites recovered from the mouse stomach 2 h after oral infection showed an extensive digestion of gp90 and increased infectivity of isolate 573, but not of isolate 587 or CL. Our data indicate that T. cruzi infection in vitro does not always correlate with in vivo infection because host factors may act on parasites, modulating their infectivity, as is the case of pepsin digestion of isolate 573 gp90.