Surprisingly fast disappearance of beta-lactam selection pressure in cultivation as detected with novel biosensing approaches

Tetracycline and beta-lactam resistances among others are used as selection markers in the production of recombinant proteins. The beta-lactam resistance is based on degradation, i.e. the selection pressure gradually disappears from the culture, whereas tetracycline resistance is based on active efflux. We have studied the kinetics of the stability of antibiotic selection pressure in culture using a simple model system (pBR322 in Escherichia coli). Concentrations of ampicillin, carbenicillin and tetracycline were measured with novel sensor cells developed in our lab. These cells are specifically induced to produce light in the presence of the drugs and here their performance was shown to be excellent in monitoring antibiotic concentrations in cell culture. The sensor cells are cheap to produce and use and a high number of samples can be analysed simultaneously. To our surprise, ampicillin and carbenicillin were completely degraded after 2.5-3.0 h of culture, although it has been widely claimed that especially carbenicillin is a good selective agent, whereas tetracycline was stable in culture. beta-lactamase activity in culture was found to correlate with the kinetics of ampicillin degradation.     

R-Ras is a global regulator of vascular regeneration that suppresses intimal hyperplasia and tumor angiogenesis

R-Ras is a small GTPase of the Ras family that regulates cell survival and integrin activity. Despite a number of in vitro studies, the in vivo function of R-Ras remains unclear. Here, we used R-Ras-null mice to explore the in vivo function of this small GTPase. Our results show a role for R-Ras as a regulator of vascular differentiation that primarily affects the remodeling of blood vessels. We show that R-Ras-null mice, although otherwise phenotypically normal, mount excessive vascular responses. We found that in vivo R-Ras expression is largely confined to fully differentiated smooth muscle cells, including those of blood vessels, and to endothelial cells. Challenging the R-Ras-null mice with arterial injury or tumor implantation showed exaggerated neointimal thickening in response to the injury and increased angiogenesis in the tumors. In wild-type mice, R-Ras expression was greatly reduced in hyperplastic neointimal smooth muscle cells and in angiogenic endothelial cells. Forced expression of activated R-Ras suppressed mitogenic and invasive activities of growth factor-stimulated vascular cells. These results establish an unexpected role for R-Ras in blood vessel homeostasis and suggest that R-Ras signaling may offer a target for therapeutic intervention in vascular diseases.     

Characterization of Actinomyces species isolated from failed dental implant fixtures

In the oral cavity, Actinomyces form a fundamental component of the indigenous microflora, being among initial colonizers in polymicrobial biofilms. However, some differences may exist between different species in terms of their attachment not only to teeth but also to biomaterials. In this study we investigated the distribution of Actinomyces in 33 dental implant fixtures explanted from 17 patients. The identification was based on comprehensive biochemical testing and gas-liquid chromatography and when needed, 16S rRNA sequencing. Actinomyces was the most prevalent bacterial genus in these failed implants, colonizing 31/33 (94%) of the fixtures. Proportions of Actinomyces growth of the total bacterial growth in the Actinomyces-positive fixtures varied from 0.01% up to 75%. A. odontolyticus was the most common Actinomyces finding, present in 26/31 (84%) Actinomyces-positive fixtures. Actinomyces naeslundii and A. viscosus were both detected in 10/31 (32%) and A. israelii in 7/31 (23%) fixtures. Other Actinomyces species, including A. georgiae, A. gerencseriae and A. graevenitzii, were detected less frequently. Our results suggest that Actinomyces species are frequent colonizers on failed implant surfaces, where A. odontolyticus was the far most prominent Actinomyces species.     

Glycosylamines of 4,6-O-butylidene-alpha-D-glucopyranose: synthesis and characterization of glycosylamines, and the crystal structure of 4,6-O-butylidene-N-(o-chlorophenyl)-beta-D-glucopyranosylamine

A total of nine glycosylamines of 4,6-O-butylidene-alpha-D-glucopyranose were synthesized using primary amines having various groups in their ortho- or para-positions. Among these, six are monoglycosylamines, including one primary glycosylamine, and three are bis-glycosylamines. All these compounds were characterized by 1H, 1H-1H COSY, 1H-13C COSY and 13C NMR spectroscopy and FTIR spectra. The FAB mass spectra provided the molecular weights of the products by exhibiting the corresponding molecular ion peaks. The crystal structure of 4,6-O-butylidene-N-(o-chlorophenyl)-beta-D-glucopyranosylamine revealed the C-1 glycosylation, the beta-anomeric nature, and the 4C1 chair conformation of the saccharide unit in the product. In the lattice two types of dimers exist. While one type of dimer is formed through O-H...O type of interactions, the other type is formed via C-H...O type of interactions. In the direction of these C-H...O type of interactions, the dimeric units are connected to form a chain.     

Comparison of cellular fatty acid profiles of the microbiota in different gut regions of BALB/c and C57BL/6J mice

The gastrointestinal tracts of developed animals are colonized by an extremely complex and diverse microbial ecosystem. The host and its microbiota are in close interaction with each other, and the hosts genetic characteristics have been suggested to have an influence on the composition of fecal bacteria. However, different sections of gastrointestinal tract harbor microbes typical of each particular section and knowledge of the effect of the hosts genotype on the microbiota in the different parts of the gastrointestinal tract is limited. In this study, mice from two inbred strains, C57BL/6J and BALB/c, were raised in identical conditions. Bacterial samples were collected from four parts of the gastrointestinal tract and analyzed for bacterial fatty acids using gas-liquid chromatography (GLC). Significant differences between the microbiota in the feces, in the cecum, in the small bowel and in the stomach were observed. Cecal samples produced more diverse bacterial fatty acid profiles than any of the other samples, revealing a higher bacterial density and a higher number of bacterial species. Further, a significant difference between the two strains of mice was observed throughout the gastrointestinal tract. These findings indicate that the hosts genotype has an influence on the gastrointestinal microbiota as a whole, and provide further evidence that the cecum is the most species-rich region of the murine gut.     

The two thrombospondin type I repeat domains of the heparin-binding growth-associated molecule bind to heparin/heparan sulfate and regulate neurite extension and plasticity in hippocampal neurons

HB-GAM (heparin-binding growth-associated molecule, also designated as pleiotrophin) and midkine form a two-member family of extracellular matrix proteins that bind tightly to sulfated carbohydrate structures such as heparan sulfate. These proteins are used by developing neurons as extracellular cues in axonal growth and guidance. HB-GAM was recently reported to enhance differentiation of neural stem cells. Based on the solution structure of HB-GAM, we have recently shown that HB-GAM consists of two beta-sheet domains flanked by flexible lysine-rich N- and C-terminal tails with no apparent structure. These domains are homologous to thrombospondin type I repeats present in numerous extracellular proteins that interact with the cell surface. Our findings showed that the two beta-sheet domains fold independently. We showed that the domains (but not the lysine-rich tails) in HB-GAM are required and sufficient for interaction with hippocampal neurons. The individual domains bind heparan sulfate weakly and fail to produce significant biological effects in neurite outgrowth and long term potentiation assays. The amino acids in the linker region joining the two domains may be replaced with glycines with no effect on protein function. These results suggest a co-operative action of the two beta-sheet domains in the biologically relevant interaction with neuron surface heparan sulfate.     

Steps toward mapping the human vasculature by phage display

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.     

Influence of hatching order on growth rate and resting metabolism of kestrel nestlings

Hatching asynchrony in altricial birds may result in a competitive disadvantage for the youngest nestlings compared to older siblings. We studied the effects of a size hierarchy on the growth rate of Eurasian kestrels Falco tinnunculus chicks in nests with and without access to supplemented food in western Finland. Body mass stopped increasing on the 19th day after hatching while body size, estimated by a combination of bone and feather lengths continued to increase at least until fledging at 26 days. Body condition, reflecting muscle and fat, did not change markedly during the growth period from the 12th day to fledging. Body temperature and resting metabolism were usually lower in nestlings 12 days old than in nestlings at fledging. Growth of body mass, size and condition, and resting metabolism were delayed in last-hatched nestlings aged 19 days. Just before fledging, last-hatched nestlings attained a similar body mass and size, and had a similar resting metabolism to those of older siblings. At fledging, only in nests without access to supplemented food was the body condition of last-hatched chicks lower than that of its siblings, but in nests with access to supplemented food no such difference was detected. Our results highlight that the level of lipids in the last-hatched nestling can be affected by the food restriction imposed by hatching order.     

Performance of Birch Seedlings Replanted in Heavily Polluted Industrial Barrens of the Kola Peninsula, Northwest Russia

Industrial barrens replacing coniferous forests around the Severonikel smelter in the Kola Peninsula, northwestern Russia, have recently expanded over 3000 ha or more. Total concentrations of metal contaminants in the upper soil layers approach 3000–5000 μg/g, and maximum hourly concentrations of sulphur dioxide in ambient air exceed 1000 μg/m³. To monitor possibilities for vegetation recovery in the denuded landscapes continuously affected by industrial emissions, we conducted several experiments with 4- to 15-year-old (1–25 cm tall) seedlings of Betula pubescens ssp. czerepanovi (mountain birch), replanted to two barren sites. Specifically, we investigated the effects of wind-sheltering, watering, and fertilization on seedling performance in the polluted sites. Sheltered and watered seedlings had more symmetrical leaves than control seedlings, suggesting less environmental stress. Consistently, sheltering and (to a lesser extent) watering improved the survival of seedlings compared with controls. The beneficial effects of watering and sheltering were most pronounced the first 2–4 weeks following planting and were greatest in the most polluted site. We conclude that the revegetation of industrial barrens can be significantly promoted by inexpensive treatments such as wind sheltering and watering, even under current emissions.     

Dose effects of transfected c-Ha-ras oncogene in transformed cell clones

We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-ras^{Val 12} oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2–4, or 4–8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the tranformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-ras^{Val 12} grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.