In vivo MRI analysis of an inflammatory injury in the developing brain.

Cerebral periventricular white matter injury stands as a leading cause of cognitive, behavioral and motor impairment in preterm infants. There is epidemiological and histopathological evidence demonstrating the role of prenatal or neonatal inflammation in brain injury in preterm infants. In order to define the effect of an inflammatory insult in the developing brain on magnetic resonance (MR) imaging, we obtained high resolution conventional and diffusion MR images of the brain of rat pups after an inflammatory injury. Rat pups were subjected on postnatal day 5 (P5) to a stereotaxic injection of lipopolysaccharide in the corpus callosum and then imaged at 11.7 T on days 0, 2 and 4 following the injury. They were subsequently sacrificed for immunohistochemistry. Diffusion tensor imaging (DTI) acquired at high spatial resolution showed an initial reduction of the apparent diffusion coefficient (ADC) in the white matter. This was followed by an increase in ADC value and in T2 relaxation time constant in the white matter, with an associated increase of radial diffusivity of the corpus callosum, and a 10-fold increase in ventricular size. On histology, these MR changes corresponded to widespread astrogliosis, and decreased proportion of the section areas containing cresyl violet positive stain. The increase in radial diffusivity, typically attributed to myelin loss, occurred in this case despite the absence of myelin at this developmental stage.

Regional assessment of atrazine exposure and incidence of breast and ovarian cancers in Kentucky.

The association between breast and ovarian cancers with endogenous estrogen or hormonally related events has led to the hypothesis that exposures to exogenous estrogenic compounds in the environment may increase the risk of these cancers. Atrazine, the most commonly used herbicide in the United States, belongs to this group of compounds and is widely used in corn production. This study is an expansion of a previous investigation conducted in Kentucky. Using secondary data, we derived several indices of environmental exposure to atrazine and examined the association between these measures and the incidence of breast and ovarian cancer in Kentucky over a 5-year period (1993-97). Exposure indices to atrazine were derived based on public water measurements, acres of corn planted, and pounds of atrazine sold. Data on breast and ovarian cancer incidence were obtained from the Kentucky Cancer Registry by county and by the 15 Area Development Districts (ADDs) in which the 120 counties are grouped. Poisson regression analyses adjusted for education and race were conducted separately for each index of exposure and for a combined total exposure score. All exposure measures were divided in quartiles for analysis. A null association was found for breast cancer across all exposure indices, both by county and by ADD. For ovarian cancer, the data suggest an inverse association, with increasing exposure linked to decreasing incidence rates, both at the county and ADD level. The following are the rate ratios (RR) and corresponding 95% confidence intervals, for the summary exposure scores in the three upper quartiles, using the lowest quartile as baseline (RR = 1.0), and the county as the unit of analysis: 1.01 (0.83-1.21), 0.77 (0.66-0.90), and 0.76 (0.65-0.88). Due to the ecologic nature of this study and inherent limitations, it is possible that other factors may be contributing to these findings. Studies using individual-level data are recommended to elucidate the relationships between estrogenic environmental exposures and female reproductive cancers.

Exogenous peptide and protein expression levels using retroviral vectors in human cells.

Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors-including those with therapeutic utility-and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 microM. We also examine other variables that contribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.

The use of modified primers to eliminate cycle sequencing artifacts.

Cycle sequencing is the workhorse of DNA sequencing projects, allowing the production of large amounts of product from relatively little template. This cycling regime, which is aimed at linear growth of the desired products, can also produce artifacts by exponential amplification of minor side-products. These artifacts can interfere with sequence determination. In an attempt to allow linear but prevent exponential growth of products, and thus eliminate artifacts, we have investigated the use of primers containing modified residues that cannot be replicated by DNA polymerase. Specifically, we have used primers containing 2'- O -methyl RNA residues or abasic residues. Oligomers consisting of six DNA residues and 20 2'- O -methyl RNA residues, with the DNA residues located at the 3'-end, primed as efficiently as DNA primers but would not support exponential amplification. Oligonucleotides containing fewer DNA residues were not used as efficiently as primers. DNA primers containing a single abasic site located six residues from the 3'-end also showed efficient priming ability without yielding exponential amplification products. Together these results demonstrate that certain types of modified primers can be used to eliminate artifacts in DNA sequencing. The technique should be particularly useful in protocols involving large numbers of cycles, such as direct sequencing of BAC and genomic DNA.

Iris color alone does not predict susceptibility to the oculocardiac reflex in strabismus surgery.

PURPOSE: To test prior published observations that patients with brown irides were more susceptible to the oculocardiac reflex. SUBJECTS AND METHODS: 466 adults and children underwent surgery on an extraocular muscle under general anesthesia without anticholinergic blockade. The first rectus muscle isolated was given a predetermined specific force pull during electrocardiograph monitoring. Iris color was retrieved from the deliberate clinical charting. RESULTS: The occurrence of the oculocardiac reflex did not correlate with age, but did profoundly for the inferior rectus and least for the lateral rectus. Iris color did not influence the incidence of occurrence of moderate or severe oculocardiac reflex. CONCLUSION: The prediction of oculocardiac reflex propensity remains elusive.

Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements.

As a means of determining whether there has been selection to conserve the basic pattern of filamentous phage mRNAs, the major mRNAs representing genes II to VIII have been defined for a phage distantly related to the Ff group specific for Escherichia coli hosts bearing F pili. Phage IKe has a genome with 55% identity with the Ff genome and infects E. coli strains bearing N pili. The results reveal a remarkably similar pattern of overlapping polycistronic mRNAs with a common 3' end and unique 5' ends. The IKe mRNAs, like the Ff phage mRNAs, represent a combination of primary transcripts and processed RNAs. However, examination of the sequences containing the RNA endpoint positions revealed that effectively the only highly conserved regulatory element is the rho-independent terminator that generates the common 3' end. Promoters and processing sites have not been maintained in identical positions, but frequently are placed so as to yield RNAs with similar coding function. By conserving the pattern of transcription and processing despite divergence in the regulatory elements and possibly the requirements for host, endoribonucleases, the results argue that the pattern is not simply fortuitous.

Functional analysis of filamentous phage f1 mRNA processing sites.

The abundant mRNAs used as templates for synthesis of filamentous phage f1 proteins are a combination of primary transcripts and 3' products of processing. The processing steps are mediated by host endoribonucleases. One of the enzymes implicated in f1 mRNA processing is RNase E, the only endonuclease thus far shown to have a global role in mRNA decay. By establishing the temperature-sensitive phenotypes of RNase E mutants and then inducing a transcription unit bearing cloned f1 processing sites, we show that RNase E is required for production of at least three of the processed RNAs. Using in vivo processing assays, we also test directly the regions implicated genetically in previous work to contain the processing sites. The sites function as discrete domains in a number of transcription units, show little influence of translation, but appear to have increased activity at the 5' terminus of an mRNA. From their functional properties, we suggest that the known processing sites from phage f1 that are dependent on RNase E may be representative of relatively late steps in rne-dependent cleavage pathways.

Binding of biotinylated DNA to streptavidin-coated polystyrene latex: effects of chain length and particle size.

The binding of 5'-end biotinylated DNA, ranging in size from 100 to 5000 base pairs, was studied using streptavidin-coated polystyrene latex particles with diameters between 0.944 and 0.090 micron. The experimental binding constants and forward rate constants of this solid-phase reaction were determined to be several orders of magnitude lower than values for the biotin-streptavidin interaction in solution as expected and were shown to depend on the size of both ligand and substrate. An observed inflection in the binding constant of the biotinylated DNA appeared around 1000 base pairs, possibly indicating different surface orientations of the macroligand above and below this critical size. This effect was more pronounced for the smaller latex particles used in this study and highlighted possible differences in the surface arrangement of streptavidin on the differently sized particles. Diffusion limitation to the binding reaction was found to be significant in all cases. In this present work, an exponential relationship was established between the experimental binding constant and the number of base pairs in the biotinylated DNA. This relationship possibly provides a means to predict capacity and binding speed in cases where adsorption, purification, and release of larger DNA chains are required.

The Minnesota Clinical Comparison and Assessment Project.

Efforts to quantify and improve the effectiveness and efficiency of health care services are critical to the health care system in the United States. Essential components of these efforts are clinician involvement and support and reliable clinical information systems. Organized medicine and the hospital industry in Minnesota have initiated and funded the Minnesota Clinical Comparison and Assessment Project (MCCAP) to begin to document, compare, and improve health care services. Through MCCAP, a forum, comprising leaders in the Minnesota health care community, oversees efforts to collaboratively identify and address quality of care. In addition, MCCAP will adapt guidelines for specific conditions/procedures, collect hospital-level data on clinical practices in the state relative to those conditions/procedures, and provide feedback so that physicians can evaluate their practice and modify it, if necessary, to improve quality of care.

Quality assurance principles for the laboratory--a brief overview.

Under the Omnibus Budget Reconciliation Act of 1986, the United States Congress has called for a study of the quality of care of Medicare beneficiaries and a strategy to assure it. The quality of laboratory services is an important component of that assessment and assurance, and hospitals need a methodology for it. The Guidelines for Quality Assessment of the Council on Medical Service of the American Medical Association can be used as a basis for a model that links structure, process, and outcome criteria to the interplay of people, equipment, and systems that comprise the laboratory. This can then support the development of a monitoring and evaluation program for its services.