Host-Parasite Interactions in Chagas Disease: Genetically Unidentical Isolates of a Single Trypanosoma cruzi Strain Identified In Vitro via LSSP-PCR

The present study aims at establishing whether the diversity in pathogenesis within a genetically diverse host population infected with a single polyclonal strain of Trypanosoma cruzi is due to selection of specific subpopulations within the strain. For this purpose we infected Swiss mice, a genetically diverse population, with the polyclonal strain of Trypanosoma cruzi Berenice-78 and characterized via LSSP-PCR the kinetoplast DNA of subpopulations isolated from blood samples collected from the animals at various times after inoculation (3, 6 and 12 months after inoculation). We examined the biological behavior of the isolates in acellular medium and in vitro profiles of infectivity in Vero cell medium. We compared the characteristics of the isolates with the inoculating strain and with another strain, Berenice 62, isolated from the same patient 16 years earlier. We found that one of the isolates had intermediate behavior in comparison with Berenice-78 and Berenice-62 and a significantly different genetic profile by LSSP-PCR in comparison with the inoculating strain. We hereby demonstrate that genetically distinct Trypanosoma cruzi isolates may be obtained upon experimental murine infection with a single polyclonal Trypanosoma cruzi strain.     

RNA-RNA interactions in the binding site of protein L24 on 23S ribosomal RNA of E. coli. II. Sequence analysis of the interacting fragments.

A ribonucleoprotein complex containing several RNA subfragments from the 5' part of 23S RNA was recovered after digestion of the reconstituted complex between 23S RNA and protein L24. It was suggested in the preceding paper that the RNA subfragments 4B, 10A and 9, which are widely separated in the sequence, strongly interact. These subfragments were previously partially sequenced by the classical fingerprinting methods. Their sequences have now been completed with rapid new RNA sequencing methods. We propose here a base-pairing model showing how these subfragments may interact with one another.     

On the isolation of TI-plasmid from Agrobacterium tumefaciens.

An efficient lysis method for Agrobacterium cells was developed, which allows a reproducible isolation of the tumor inducing (TI)-plasmid. The lysis method is based on the sensitivity of this bacterium to incubation with lysozyme, n-dodecylamine,EDTA, followed by Sarkosyl, after growth in the presence of carbenicillin. We also present a procedure for the isolation of the TI-plasmid on a large scale, that might be used for the mass isolation of other large plasmids which like the TI-plasmid, can not be cleared with earlier described procedures. The purity of the plasmid preparations was determined with DNA renaturation kinetics, which method has the advantage that the plasmid need not to be in the supercoiled or open circular form.     

Probability graphs resolve components with buoyant-density differences of 0.001 g/cm3 in equilibrium density gradients

A method derived from DNA melting-curve analysis has been modified for determining homogeneity of DNA species centrifuged to equilibrium in a CsCl gradient. DNA species of known density were purified, centrifuged to equilibrium in a Spinco Model E analytical ultracentrifuge, and analyzed by probability distribution. In order to determine the limits of resolution by this procedure, samples with varying buoyant-density separations were obtained by mixing different DNA species. Results indicate this method can detect overlapping mixtures of DNA in relative amounts of 9:1 and resolve two species of DNA of equal concentrations with a buoyant-density separation of 0.001 g/cm³.     

Increased gene expression of plasminogen activators and inhibitors in left ventricular hypertrophy

In the early stages of left ventricular hypertrophy (LVH) acute adaptive changes occur in the coronary vasculature as it remodels. Plasminogen activators (PAs) and inhibitors (PAIs) have the potential effects of proteolytic degradation that is relevant to tissue remodeling and angiogenesis. Our study focused on the possible roles of PAI-1, PAI-2, uPA and tPA in myocyte hypertrophy and angiogenesis in the early and late stages of pressure overload induced left ventricular hypertrophy (LVH). We divided seventeen adult swine, weighing 24.2 ± 6.5 kg, into four groups: control, sham-operated, early LVH and late heart failure LVH group. At surgery we placed a fixed constrictor on the ascending aorta immediately above the aortic valve. This increased LV systolic pressure from 133 ± 15 to 193 ± 24 mm Hg after the surgery. We subdivided the early group into groups of 3 animals each that we euthanized at 8, 24 and 72 h after operation and obtained heart samples for analysis. In the late heart failure group individual animals were euthanized at 55, 59, 62 and 72 days after the detection of congestive heart failure. We also obtained tissue samples from the control and sham-operated swine. Sections for histologic analysis were fixed in 10% buffered formalin. We isolated RNA, size fractionated it using 1% formaldehyde-agarose gel electrophoresis and then did Northern blots. The mRNAs from both PAI-1 and PAI-2 showed a remarkable increase at 8 and 24 h after acute aortic constriction and returned to control by 72 h. Regional differences showed that most of the increases were in the endocardium. Three animals in the late heart failure LVH group were determined to be in congestive heart failure at about 2 months after the onset of aortic constriction. In these animals PAI-1 and PAI-2 were increased in both the left and right ventricles but remained low in an animal of the same elevation in aortic pressure seen by the LV who did not have congestive failure. These data suggest that PA and PAI gene expressions change before morphologic changes occur in the early stages of developing LVH. Also at the time of onset of congestive heart failure this increased expression reappears. PAs and PA inhibitors mRNA levels vary in the different regions of the heart reflecting changing wall stresses. Thus, the PAs and PA inhibitors may play an important role in angiogenesis that occurs during the early stages of LVH. The increased expression in the late stage of LVH may reflect further changes in wall stresses since these animals also showed overt clinical signs of heart failure.     

Adenylate Energy Charge in Escherichia coli During Growth and Starvation

The value of the adenylate energy charge, [(adenosine triphosphate) + (1/2) (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types.