Uptake and killing of Listeria monocytogenes by normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting

Cellular components of innate immunity (NK cells, monocytes and granulocytes) play an important role in early resistance to Listeria monocytogenes in the mouse model. Minimally invasive methods of measuring the bacteriocidal capacity of these cells may be useful as a biomarker of susceptibility in humans. A technique was developed whereby the uptake and survival of L. monocytogenes could be measured in human granulocytes and monocytes using small volumes of peripheral blood. This method used flow cytometry to detect the presence of PKH-2-labeled bacteria within these cells. Survival of bacteria was determined by sorting of infected cells based on a combination of fluorescence and light scattering properties. Considerable variation in bacterial recovery was seen between normal volunteers. There was consistently greater survival of a fully virulent strain of L. monocytogenes within monocytes and granulocytes compared with an isogenic strain lacking the hemolysin, listeriolysin O, when measured at baseline. There was no evidence of longer-term bacterial survival or growth at 2 or 24 h. This technique may be useful for assessment of both host resistance and pathogen virulence.     

Fed-batch cultivation of Pseudomonas cepacia with on-line control of the toxic substrate salicylate

Pseudomonas cepacia was cultivated on salicylate as sole carbon and energy source in a fed-batch culture. On-line measurement of salicylate concentration was carried out using a filtration system. Cell-free permeate was passed through a flow-through spectrophotometer. Measurements were carried out at 325 nm. A Proportional-Integral controller was used for regulating the feed rate around a basic dosage scheme to maintain a constant salicylate concentration of 0.2 g/l in the broth. The cell mass concentration increased from less than 1 g dry weight (dw)/l to 12 g dw/l in less than 6 h, and the yield coefficient was 0.4 g dw/g salicylate. The intracellular enzymatic activity of salicylate hydroxylase was virtually unchanged during the fed-batch cultivation. Correspondence to: A. Tocaj     

Structural analysis of denaturant-protein interactions: Comparison between the effects of bromoethanol and SDS on denaturation and renaturation of triclinic lysozyme

This paper summarizes our crystallographic studies of the interaction of denaturants with cross-linked triclinic lysozyme. Electron density maps of various bromoethanol-lysozyme complexes are analyzed and compared to those reported earlier for SDS-lysozyme complexes. Despite differences in the chemical nature and size of the two denaturants their mode of interaction with the protein is quite similar, suggesting the existence of a general mechanism for binding of hydrophobic-hydrophilic denaturants to proteins. Our results are consistent with the conclusion that lysozyme consists of two domains connected by a flexible segment and that this segment represents an internal degree of freedom of the protein.The work was carried out during the tenure of a fellowship from the European Molecular Biology OrganizationWe are grateful to Dr. Gerson Cohen for providing us with his data processing programs, to Drs. David Haas, Paul Sigler, Thomas Creighton and Micael James for helpful discussions, and to Mr. Samuel Getteno for his invaluable technical assistance.     

Granular pneumocytes and altitude: A stereological evaluation

Summary Stereological analysis demonstrates that the granular pneumocytes of mice native to high altitude (4660 m) are significantly larger than those of the same species (Phyllotis darwini) living at sea level. Such cells have larger nuclei and a significantly greater volume of mitochondria. There are both more and larger lamellar bodies in high altitude granular pneumocytes, resulting in about four times more surfactant per cell at high altitude.     

Biosynthesis and secretion of tropoelastin by chick aorta cells

Cells were isolated from the aortae of 17-day old chick embryos by digestion of the vessels with a combination of trypsin and collagenase. When these cells were incubated in suspension culture in Krebs-Ringer media containing pancreatic trypsin inhibitor and radioactive amino acids, they synthesized and secreted labeled proteins into the media. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the secreted proteins labeled with [¹⁴C]proline revealed two major components. The larger component with an approximate molecular weight of 125,000 had a [¹⁴C]hydroxyproline content consistent with a form of procollagen. The molecular weight of 70,000 and [¹⁴C]hydroxyproline content of the second component was consistent with that previously reported for tropoelastin extracted from chick aortae. By following the kinetics and secretion of tropoelastin labeled with [³H]valine, we have estimated that 17 minutes are required to synthesize and secrete the molecule under these experimental conditions.     

Development during single IVP of bovine oocytes from dissected follicles: Interactive effects of estrous cycle stage,follicle size and atresia

Previous work suggests that a number of factors such as follicle size, day of estrous cycle, and level of atresia influence the developmental potential of bovine oocytes in vitro. To understand better the interactions of these factors, 1299 follicles ≥3 mm in diameter were dissected from ovaries of synchronized dairy cows on four days (d2, d7, d10, or d15) during the estrous cycle. The oocyte from each follicle was collected and matured, fertilized, and cultured singly to d8 (d0 of culture = IVF). Control follicles (302) were similarly dissected and processed from an ovary pair randomly collected from the abattoir on each slaughter day. Results showed that development to blastocyst was greater in oocytes collected during phases of follicular growth (d2 and d10) than those collected during phases of follicular dominance (d7 and d15; 44.8% vs. 36.0%, respectively: P < 0.001) over all follicle size categories (3–5 mm, 6–8 mm, 9–12 mm and ≥13 mm). Oocyte competence tended to increase with increasing follicle size (P < 0.1). Follicular cells from follicles containing an oocyte that developed to morula or greater by d8 (484 samples) were analyzed by flow cytometry to measure the level of apoptosis. Results showed an increase in mean percent apoptotic cells in subordinate follicles (18.65 ± 0.86 over all size categories), particularly those of medium size (25.55 ± 2.2 for 6–8 mm size follicles; P < 0.001), during the dominance phase compared to growth phase (9.25 ± 0.95 over all sizes; P < 0.05). These results show a significant affect of the stage of estrous cycle on both oocyte competence and levels of follicular atresia. Mol. Reprod. Dev. 53:451–458, 1999. © 1999 Wiley‐Liss, Inc.     

Population genomics and geographic dispersal in Chagas disease vectors: Landscape drivers and evidence of possible adaptation to the domestic setting

Accurate prediction of vectors dispersal, as well as identification of adaptations that allow blood-feeding vectors to thrive in built environments, are a basis for effective disease control. Here we adopted a landscape genomics approach to assay gene flow, possible local adaptation, and drivers of population structure in Rhodnius ecuadoriensis, an important vector of Chagas disease. We used a reduced-representation sequencing technique (2b-RADseq) to obtain 2,552 SNP markers across 272 R. ecuadoriensis samples from 25 collection sites in southern Ecuador. Evidence of high and directional gene flow between seven wild and domestic population pairs across our study site indicates insecticide-based control will be hindered by repeated re-infestation of houses from the forest. Preliminary genome scans across multiple population pairs revealed shared outlier loci potentially consistent with local adaptation to the domestic setting, which we mapped to genes involved with embryogenesis and saliva production. Landscape genomic models showed elevation is a key barrier to R. ecuadoriensis dispersal. Together our results shed early light on the genomic adaptation in triatomine vectors and facilitate vector control by predicting that spatially-targeted, proactive interventions would be more efficacious than current, reactive approaches.