Two-dimensional display and whole genome comparison of bacterial pathogen genomes of high G+C DNA content

Three highly mutable loci of the wall-less pathogens Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae undergo high-frequency genomic rearrangements and generate extensive antigenic variation of major surface lipoproteins. Adjacent to each locus, an open reading frame exists as a single chromosomal copy and is predicted to encode a site-specific DNA recombinase exhibiting high homology to the recombinases XerD of Escherichia coli and CodV of Bacillus subtilis. Each of the mycoplasmal proteins are members of the lambda integrase family of tyrosine site-specific recombinases and likely mediates site-specific DNA inversions observed within the adjacent, variable loci.     

A structural model for the catalytic cycle of Ca(2+)-ATPase

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.     

The role of IFN in respiratory syncytial virus pathogenesis

Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1(-/-) mice. STAT1(-/-) mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1(-/-) animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-gamma(-/-)-infected mice. In cytokine analyses of infected lung tissue, IFN-gamma was induced in both STAT1(-/-) and wild-type mice, with preferential IL-4, IL-5, and IL-13 induction only in the STAT1(-/-) animals. Eotaxin was detected in the lungs of both wild-type and STAT1(-/-) mice following infection, with a 1.7-fold increase over wild-type in the STAT1(-/-) mice. Using a peptide epitope newly identified in the RSV fusion protein, we were able to demonstrate that wild-type memory CD4(+) T cells stimulated by this peptide produce primarily IFN-gamma, while STAT1(-/-)CD4(+) cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I (alphabeta) and type II (gamma) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.     

Use of lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters

AIMS: Use of lacticin 481 to facilitate the conjugal transfer of the bacteriophage resistance plasmid pCBG104 to various starter cultures. METHODS AND RESULTS: A raw milk isolate of Lactococcus was found to harbour determinants for lacticin 481 production and immunity and phage resistance on a plasmid designated pCBG104. The lacticin 481 was successfully used to mobilize the phage resistance determinant to a variety of cheese starters enabling the formation of highly phage resistant starters. In addition, it facilitated the stacking of a number of phage resistance genes, namely a type I restriction modification system, a phage abortive infection system and a phage adsorption blocking system in a single Lactococcus strain without the use of recombinant techniques. The transconjugants were all shown to produce lacticin 481 and to contain the entire 481 operon. Subsequently one transconjugant was selected and successfully used for large-scale cheddar cheese manufacture. CONCLUSIONS: Lacticin 481 could be used as a food-grade selectable marker to facilitate the introduction of advantageous traits to starter cultures for industrial food fermentations. SIGNIFICANCE AND IMAPCT OF THE STUDY: Food-grade selectable markers greatly facilitate the introduction of various advantageous traits to starter cultures for industrial food fermentation. Indeed self-cloning which is becoming increasingly important for strain improvement has a requirement for the identification and demonstration of the utility of tools such as lacticin 481.     

Changing Exploitation of Terrestrial Vertebrates During the Past 3000 Years on Tobago, West Indies

On the southern Caribbean island of Tobago, we excavated two archaeological deposits, the preceramic (ca. 2900 years old) Milford 1 site (TOB-3) and the ceramic (ca. 1200 to 900 years old) Golden Grove site (TOB-13). The non-fish bone assemblages from these sites are very different, with an emphasis at TOB-3 on sea turtles (which seldom nest today on Tobago) and the collared peccary Tayassu tajacu, a large (17–30 kg) mammal extremely rare now on Tobago. TOB-3 also yielded bones of two somewhat smaller mammals that are extinct on Tobago, the red howler monkey Alouatta seniculus and paca Agouti paca. The non-fish species at TOB-3 comprise only four reptiles and seven mammals, whereas they are more diverse (29 species) at TOB-13 and mostly represent small- to medium-sized vertebrates (<10 kg, often <1 kg), such as a toad, lizards, snakes, birds, opossum, armadillo, and eight species of rodents, as well as sea turtle and peccary, although the latter is uncommon. We interpret these differences as possibly being due to a local or island-wide scarcity of big game (peccaries) in ceramic times, which would have promoted diversification of hunting practices. Related to this may have been a more sedentary way of life for Tobago's ceramic peoples, with increased agriculture leading to more hunting near the site and less time being devoted to longer distance big-game hunting. While prehistoric anthropogenic extinction or population reduction of vertebrates is well documented on Caribbean islands to the north, our data from Tobago show that such depletions probably occurred as well on continental, land-bridge islands with more diverse faunas. Models of post-Pleistocene faunal relaxation toward a lower value for species richness should not ignore human-caused losses, which may be impossible to distinguish from non-anthropogenic losses.     

DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.     

Secular trend in body height and weight of Australian children and adolescents

Secular changes in growth and maturation have been well documented in various world populations, with secular increase especially noticeable in the developed countries. To assess the trend in both adult size and tempo of growth we compared the data on stature and body weight obtained in 1992-1993 from 1,804 Melbourne school students aged 5 to 17 with historical data collected from white Australians during the last 100 years. We illustrate the age-dependent trend in stature and body weight by means of regression surfaces. These were constructed by fitting local regression models to historical data and by simple plots showing the overall, and per decade, secular increase in both these measures at peripubertal and adult ages. Because of limited information on sample sizes and variability provided by the historical data, statistical comparisons have been performed only between the present 1992-1993 survey and two earlier independent surveys conducted in 1985 and 1970. The results have shown secular increase in adult stature over the last century, with the rate of increase varying between 0.4 and 2.1 cm/decade in males and 0.01 and 1.6 cm/decade in females. While secular increase in stature has significantly slowed down during the last two decades, the increase in body weight is still continuing at a high rate, and this increase is more pronounced in females. The period of strong secular increase, especially in the tempo of growth, coincided both with the shift toward earlier menarche and the improvement of socioeconomic conditions of the Australian population. The need for further studies to identify factors determining the continuing increase in body weight is emphasized, and caution in using the existing national growth standards for stature and weight is recommended.     

Rapid reversibility of the allergen-induced pulmonary late-phase reaction by an intravenous beta 2-agonist

This studywas performed to determine the degree to which₂-adrenergic receptor agonistscan reverse the allergen-induced late reduction in lungfunction. On two occasions, seven asthmatic subjects wereadministered terbutaline or its vehicle by intravenous infusion 7 hafter inhaled allergen, at which point the forced expiratory volume in1 s was 57% of baseline. On another occasion, terbutaline was infusedat baseline to determine maximal attainable bronchodilation. Afterallergen challenge, terbutaline rapidly improved lung function. At theend of terbutaline infusion, the forced expiratory volume in 1 sreached 100 ± 1.3% of baseline and 84.2 ± 4.3% of maximalattainable value, but the bronchodilating effect of the -agonist didnot plateau. The values for forced vital capacity were 102 ± 1.3%of baseline and 95.1 ± 3% of maximal attainablevalue. The kinetics of the terbutaline effect, when it wasinfused at baseline, were similar to those in the late phase. Becausethe late-phase reduction in lung function is rapidly reversible by₂-adrenergic agonists, weconclude that it is caused mainly by bronchial smooth muscle spasm.

     

Neuroblastoma Tyrosine Kinase Signaling Networks Involve FYN and LYN in Endosomes and Lipid Rafts

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.