Genetic and Physical Mapping of the Mcra (Rgla) and Mcrb (Rglb) Loci of Escherichia Coli K-12

We have genetically analyzed, cloned and physically mapped the modified cytosine-specific restriction determinants mcrA (rglA) and mcrB (rglB) of Escherichia coli K-12. The independently discovered Rgl and Mcr restriction systems are shown to be identical by three criteria: 1) mutants with the RglA- or RglB- phenotypes display the corresponding McrA- or McrB- phenotypes, and vice versa; 2) the gene(s) for RglA and McrA reside together at one locus, while gene(s) for RglB and McrB are coincident at a different locus; and 3) RglA+ and RglB+ recombinant clones complement for the corresponding Mcr-deficient lesions. The mcrA (rglA) gene(s) is on the excisable element e14, just clockwise of purB at 25 min. The mcrB (rglB) gene(s), at 99 min, is in a cluster of restriction functions that includes hsd and mrr, determinants of host-specific restriction (EcoK) and methyladenine-specific restriction respectively. Gene order is mcrB-hsdS-hsdM-hsdR-mrr-serB. Possible models for the acqusition of these restriction determinants by enteric bacteria are discussed.     

Modified inoculum for radiometric susceptibility testing ofMycobacterium tuberculosis

Two-hundred and fifteen isolates ofMycobacterium tuberculosis were evaluated with the BACTEC 460 radiometric method for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin (SM); a revised protocol for inoculum preparation was used. Fresh clinical isolates were subcultured into 7H9 broth and then photometrically adjusted to the equivalent of a 0.5 McFarland standard, one-half the recommended inoculum density. This method produced an overall 98.3% correlation with a conventional agar method. The sensitivity of this procedure was good for all drugs tested except for the lowest concentration of SM (2 g/ml). Specificity was excellent for all drugs tested. After repeat testing, only four discrepancies were found, yielding a 99.8% correlation between the two systems. The time required for susceptibility tests averaged 4.6 days. This method for inoculum preparation effectively minimized the number of susceptibility tests exceeding the threshold value before the fourth day of incubation. This allowed for definite trends of the growth index values to become established before interpretation of results.     

Resting cortisol levels and the emergence of dominant status among male vervet monkeys

Resting serum cortisol was measured in adult male vervet monkeys (Cercopithicus aethiops sabaeus) in four different conditions: (1) among groups with unaltered group membership and established dominance hierarchies; (2) among groups from which the original dominant male had been removed and in which the remaining males competed for dominant status; (3) among newly formed groups of three unfamiliar males each of which had been the dominant male in his previous group; and (4) among groups from which a dominant male was temporarily separated and returned. In Condition 1, cortisol concentrations did not differ between dominant and subordinate males. The second condition showed that cortisol levels were highest among males who eventually emerged as the dominant male. In the third condition, however, cortisol levels did not differentiate eventually dominant from eventually subordinate males. In the last condition, cortisol levels were highest in the animals that became or remained dominant following reintroduction. These data indicate that cortisol concentration does not differ between dominant and subordinate males in stable groups and that cortisol rises during competition for dominance among familiar males.     

13C NMR spectroscopy of Methanobacterium thermoautotrophicum. Carbon fluxes and primary metabolic pathways

The flux of 13C-labeled carbons from the soluble metabolite 2,3-cyclopyrophosphoglycerate (CPP), a novel compound found in high concentrations exclusively in methanobacteria and methanobrevibacter, into carbohydrate-containing material has been deduced by solid-state 13C NMR spectroscopy which strongly argues for a role in gluconeogenesis for this unique metabolite. The turnover rates, but not the steady-state levels, of CPP labeled by 13CO2 or [13C]acetate depend dramatically on cell growth conditions. When the demand for carbohydrate synthesis is reduced (i.e. in stationary phase), the rates of CPP biosynthesis and degradation decrease 10-fold, and the disaccharide alpha, alpha-trehalose accumulates. Valinomycin, a metabolic inhibitor of Methanobacterium thermoautotrophicum growth, does not affect steady-state levels of CPP, but does decrease 13C uptake into the CPP pool. The effects of these different conditions on CPP labeling suggest stringent regulation of CPP linked to cellular metabolism. Labeling of CPP by [6-(13)C]glucose, which does not serve as an energy or carbon source for this organism, provides strong evidence that glucose is cleaved by the reverse of the gluconeogenesis pathway. This metabolic pathway linking glucose with triose phosphate type precursors and an analysis of the 13C NMR spectrum of CPP labeled by incubating cells with [U-13C]glucose have established that in vivo phosphoenolpyruvate synthetase must be reversible.     

Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate.

Two opioid peptides were isolated from a bovine hemoglobin hydrolysate, by use of gel permeation (GP) and reverse phase (RP) high performance liquid chromatography (HPLC). Their primary structure and accurate molecular weights, determined by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, were identical to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin 7) of the beta-chain of bovine hemoglobin. The same fragments occur in human hemoglobin in positions 32-41 and 33-41 of the beta-chain, respectively. The opioid potency of these peptides, exhibited by use of electrically stimulated muscle of isolated guinea-pig ileum (GPI), were significant and comparable with some others previously described. In addition, the location of the two opioid peptides, VV-hemorphin-7 and LVV-hemorphin-7, revealed the existence of a "strategic zone" both in the bovine and human beta-chains of hemoglobin.     

Polymorphic Admixture Typing in Human Ethnic Populations

A panel of 257 RFLP loci was selected on the basis of high heterozygosity in Caucasian DNA surveys and equivalent spacing throughout the human genome. Probes from each locus were used in a Southern blot survey of allele frequency distribution for four human ethnic groups: Caucasian, African American, Asian (Chinese), and American Indian (Cheyenne). Nearly all RFLP loci were polymorphic in each group, albeit with a broad range of differing allele frequencies (δ). The distribution of frequency differences (δ values) was used for three purposes: (1) to provide estimates for genetic distance (differentiation) among these ethnic groups, (2) to revisit with a large data set the proportion of human genetic variation attributable to differentiation within ethnic groups, and (3) to identify loci with high δ values between recently admixed populations of use in mapping by admixture linkage disequilibrium (MALD). Although most markers display significant allele frequency differences between ethnic groups, the overall genetic distances between ethnic groups were small (.066–.098), and <10% of the measured overall molecular genetic diversity in these human samples can be attributed to “racial” differentiation. The median δ values for pairwise comparisons between groups fell between .15 and .20, permitting identification of highly informative RFLP loci for MALD disease association studies.     

How do floater male eastern bluebirds benefit by filling vacancies on territories where females already have young?

When male eastern bluebirds (Sialia sialis) are killed or deserttheir mates, replacement males usually take over the territory.We captured 25 eastern bluebird males and held them in captivityto study the behavior of replacement males and their responseto young already present in the nest. Of 17 replacement males,most courted the females, and 23% of experimental females renested,a proportion not different from that of control females. Noreplacement males were infanticidal, and none fully adoptedthe young. Replacement males spent significantly less time onthe territory and defended it significantly less than controlmales. We discuss breeding options available to a floater maleeastern bluebird and conclude that (1) floater male bluebirdsthat fill territory vacancies benefit by gaining breeding opportunitiesin the current breeding season, and (2) indifference to theyoung is the least costly option for replacement male easternbluebirds.     

Defibrotide inhibits donor leucocyte‐endothelial interactions and protects against acute graft‐versus‐host disease

Allogeneic hematopoietic stem cell transplantation (allo‐HCT) is an effective therapy for the treatment of high‐risk haematological malignant disorders and other life‐threatening haematological and genetic diseases. Acute graft‐versus‐host disease (aGvHD) remains the most frequent cause of non‐relapse mortality following allo‐HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi‐target drug successfully employed for prophylaxis and treatment of veno‐occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo‐HCT. Using a fully MHC‐mismatched murine model of allo‐HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD‐associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo‐HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte‐endothelial interactions by down‐regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo‐HCT, paving the way for new therapeutic approaches.