A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.     

Contribution of neutral processes to the assembly of gut microbial communities in the zebrafish over host development

Despite their importance to host health and development, the communities of microorganisms associated with humans and other animals are characterized by a large degree of unexplained variation across individual hosts. The processes that drive such inter-individual variation are not well understood. To address this, we surveyed the microbial communities associated with the intestine of the zebrafish, Danio rerio, over developmental time. We compared our observations of community composition and distribution across hosts with that predicted by a neutral assembly model, which assumes that community assembly is driven solely by chance and dispersal. We found that as hosts develop from larvae to adults, the fit of the model to observed microbial distributions decreases, suggesting that the relative importance of non-neutral processes, such as microbe-microbe interactions, active dispersal, or selection by the host, increases as hosts mature. We also observed that taxa which depart in their distributions from the neutral prediction form ecologically distinct sub-groups, which are phylogenetically clustered with respect to the full metacommunity. These results demonstrate that neutral processes are sufficient to generate substantial variation in microbiota composition across individual hosts, and suggest that potentially unique or important taxa may be identified by their divergence from neutral distributions.     

Reducing accident death rates in children and young adults: the contribution of hospital care. Steering Committee of the Major Trauma Outcome Study Group.

OBJECTIVE: To assess the contribution of trauma care to the recent decline in accident death rates among children and young people. DESIGN: Logistic regression modelling of temporal trends in the probability of death in patients admitted to hospital for the treatment of severe injury. SETTING: Hospitals participating the United Kingdom major trauma outcome study. SUBJECTS: 3230 patients with an injury severity score of 16 or more, who were admitted for more than three days, transferred or admitted to intensive care, or died from their injuries. MAIN OUTCOME MEASURES: Death or survival in hospital within three months of injury. RESULTS: Over the seven year period 1989-95 there was a substantial decline in the probability of death among children and young adults admitted to hospital after severe injury. The overall estimate of the reduction in the odds of death was 16% per year (odds ratio for the yearly trend 0.84; 95% confidence interval 0.79 to 0.89). This decline did not differ significantly between age groups. (0-4 years 0.79; 5-14 years 0.87; 15-24 years 0.83). CONCLUSIONS: Reductions in hospital case fatality have made an important contribution to reaching the Health of the Nation targets. The contribution of hospital care in the reduction of accident mortality should be taken into account in decisions about the allocation of resources to preventive and curative services.     

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.     

Fish and macroinvertebrate assemblages reveal extensive degradation of the world's rivers

Rivers suffer from multiple stressors acting simultaneously on their biota, but the consequences are poorly quantified at the global scale. We evaluated the biological condition of rivers globally, including the largest proportion of countries from the Global South published to date. We gathered macroinvertebrate- and fish-based assessments from 72,275 and 37,676 sites, respectively, from 64 study regions across six continents and 45 nations. Because assessments were based on differing methods, different systems were consolidated into a 3-class system: Good, Impaired, or Severely Impaired, following common guidelines. The proportion of sites in each class by study area was calculated and each region was assigned a Köppen-Geiger climate type, Human Footprint score (addressing landscape alterations), Human Development Index (HDI) score (addressing social welfare), % rivers with good ambient water quality, % protected freshwater key biodiversity areas; and % of forest area net change rate. We found that 50% of macroinvertebrate sites and 42% of fish sites were in Good condition, whereas 21% and 29% were Severely Impaired, respectively. The poorest biological conditions occurred in Arid and Equatorial climates and the best conditions occurred in Snow climates. Severely Impaired conditions were associated (Pearson correlation coefficient) with higher HDI scores, poorer physico-chemical water quality, and lower proportions of protected freshwater areas. Good biological conditions were associated with good water quality and increased forested areas. It is essential to implement statutory bioassessment programs in Asian, African, and South American countries, and continue them in Oceania, Europe, and North America. There is a need to invest in assessments based on fish, as there is less information globally and fish were strong indicators of degradation. Our study highlights a need to increase the extent and number of protected river catchments, preserve and restore natural forested areas in the catchments, treat wastewater discharges, and improve river connectivity.     

The binding of nucleotides and bivalent cations to the calcium-and-magnesium ion-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum.

The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.     

Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases

1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (alpha) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to alpha for NADH binding to give the alcohol dehydrogenase-NADH-isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase-NADH complex and the bovine glutamate dehydrogenase-NADH-glutamate complex. The non-linearity is accurately represented by F=[1-alpha(1-x)](n) where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and alpha. 4. The prediction from the equation F=[1-alpha(1-x)](n) that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when alpha=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.     

New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks

Summary Methods are described for the direct cytochemical demonstration of the enzymes nonspecific esterase and acid phosphatase based on synthetic substrates which initially deposit Hatchett's brown (cupric ferrocyanide, Cu₂Fe(CN)₆·7 H₂O) at their subcellular sites. The small amounts of Hatchett's brown deposited as a result of the enzyme's activity may be intensified by bridging to osmium through thiocarbohydrazide. Alternatively, even greater amplification of the sites of activity may be attained by utilizing the Hatchett's brown as a catalyst to effect the oxidative coupling of 3,3-diaminobenzidine resulting in the formation of an osmiophilic indamine-type polymer.One of the major advantages of this new approach is that it permits the study of acid hydrolase localization without lead in the incubation medium. Studies were performed with these methods having identical incubation media except for synthetic substrate in many different cell types and tissues. They verify a frequent nonlysosomal localization for acid phosphatase and the heterogeneity of lysosomes and lysosomal populations with respect to hydrolase content.These methods give information obtained by direct cytochemical observation an advantage not previously held, in comparison with information from cell-fractionation cytochemical or biochemical studies. Initial studies with these methods on many tissues reinforce previous suggestions of the involvement of acid hydrolases in extralysosomal sites in subcellulur anabolic processes.Supported by U.S.P.H.S. Grant DE-02668.Dr. Anderson's work was performed at the Department of Anatomy of the University of Chicago and was supported by Research Grant No. M 71-077C from the Population Council.     

Acetylene reduction with physiological electron donors by extracts and particulate fractions from nitrogen-fixing Azotobacter chroococcum

1. 1. Preparations were obtained from Azotobacter chroococcum which reduced acetylene to ethylene using physiological electron donors instead of sodium dithionite. These preparations fell into two categories: those which required catalytic amounts of benzyl viologen for acetylene reduction and those that did not.

2. 2. Acetylene reduction without benzyl viologen or sodium dithionite was observed only with particles that sedimented at 40 000 × g after disrupting bacteria in the French press or with preparations obtained by disrupting bacteria protected by a mixture of defatted bovine serum albumin-Ficoll-MgCl₂ with liquid N₂; supernatant fractions required benzyl viologen for acetylene reduction.

3. 3. Added ATP inhibited acetylene reduction by large particles; ATP and MgCl₂ were necessary for maximum acetylene reduction with bovine serum albumin-protected preparations.

4. 4. NADH and carbon substrates acted as electron donors but H₂ did not; NAD⁺ was necessary for maximum acetylene reduction with carbon substrates.

5. 5. Anaerobic conditions were necessary for maximum acetylene reduction in all cases.