Development of a human DNA quantitation system.

The AluQuant Human DNA Quantitation System has been developed for human-specific quantitation of forensic samples. This system uses probes specific to repetitive genetic elements allowing quantitation without target amplification. Target immobilization is unnecessary with employment of solution hybridization. The AluQuant Human DNA Quantitation System uses a series of enzymatic reactions to produce a luminescent signal proportional to the quantity of human DNA present. This report demonstrates a range of quantitation from 0.1-50 ng of human DNA. Signal from non-human DNAs tested was insignificant and addition of non-human DNAs into a human sample did not alter quantitation. Lastly, the system was unaffected by degradation of sample through sonication. The AluQuant Human DNA Quantitation System is a simple and sensitive method for quantitating the concentration of human DNA in forensic samples.

A bioluminescent Escherichia coli auxotroph for use in an in vitro lysine availability assay.

Microbiological methods have been used to determine the amino acid availability of a variety of animal feed and human food protein sources. Growth of Escherichia coli auxotrophs have been shown to yield a consistent linear response to lysine concentration when compared to chemical measures. Extent of total growth of E. coli lysine mutant (American Type Culture Collection #23812) when measured as optical density (OD) displays a lysine-dependent growth response that can be used to estimate lysine in feed proteins. However, typical OD-based growth studies for amino acid quantitation using the mutant may require anywhere from 12 to over 40 h. To develop an improved rapid method for lysine quantitation in protein sources, the plasmid pJHD500 carrying genes that encode for expression of bioluminescence and ampicillin resistance was transformed into the E. coli mutant by electroporation (set at 1.80 kV). The luminescence measured during early exponential growth allowed detectable differentiation of lysine concentration in the media in 4 h. When the luminescence method was compared with the conventional optical density lysine growth assay, the correlation coefficient was 0.989. Lysine availability valued for enzymatically hydrolyzed protein sources were comparable with availability measures using animal methods for lysine availability. This research shows potential applications for more rapid quantitative measurement of bioavailable lysine.

Antibiotic amendment for suppression of indigenous microflora in feed sources for an Escherichia coli auxotroph lysine assay.

Growth responses of lysine auxotrophic mutants of Escherichia coli have been used as a measurement of bioavailable lysine in protein sources and animal feeds. Sterilizing feed samples by autoclaving to eliminate non-specific background growth of indigenous feed micro-organisms prior to conducting the bacterial assay may introduce chemical and physical alterations to the feeds, influencing the estimation of available feed lysine. In this study, an antibiotic- and antifungal-supplemented medium was constructed to support growth of an E. coli lysine auxotroph assay organism, and was tested for its ability to repress indigenous bacterial and fungal growth in feed samples. To determine which antibiotics to include, an ampicillin-sensitive E. coli lysine mutant strain (ATCC no. 23812) was screened for antibiotic resistance and transformed with a plasmid carrying an ampicillin resistance gene. Maximum optical density quantitative response of the E. coli auxotroph to lysine was not altered by the antibiotic medium amendments (ampicillin, novobiocin and cycloheximide). Indigenous microfloral growth in a variety of typical animal feeds was suppressed in the presence of the antistatic agents. The estimated lysine recovery was 91.6% and 98.1% when the medium was used in an assay of available lysine in a lysine-supplemented feed. This indicates that the antibiotic-amended basal medium can be used for the E. coli-determined lysine availability of a variety of animal feeds without prior sterilization of the feed sources.

The progression of morphologic changes in bronchopulmonary dysplasia.

Review of published reports shows confusion regarding the pathologic sequelae of neonatal respiratory distress. To examine this problem the authors studied histologic slides of lung from 46 patients so diagnosed listed in the autopsy files of The Johns Hopkins Hospital. Two distinct morphologic patterns emerged. In 26 patients (Group 1) there were varying sized areas of interstitial fibrosis with associated distortion of air spaces. The process was nonspecific and closely resembled the interstitial fibrosis of varying etiologies found in adults. This lesion appears to correspond to most descriptions of bronchopulmonary dysplasia. A second process predominated in 10 patients (Group 3). There were normal conducting bronchi, marked uniform enlargement of distal air spaces, and little or no interstitial fibrosis. In 10 patients (Group 2) both lesions coexisted. To gain further insight into the morphology of these disorders, the authors reconstructed serial histologic sections of lung from three infants of varying sizes with normal lungs and infants of varying ages with bronchopulmonary dysplasia. The results confirmed the observations made on routine histologic sections by showing interstitial fibrosis in the early stages of bronchopulmonary dysplasia and a reduced number of very large terminal air spaces without interstitial fibrosis in the late stages. There were no obvious differences in the clinical courses of infants with the different morphologic stages; but Group 1 patients averaged 39 days of age, Group 2 lived 142 days, and Group 3 survived 277 days. It seems probable that early bronchopulmonary dysplasia is simply the healing of alveolar wall injury of whatever cause, most commonly hyaline membrane disease of the newborn; and that in the later phases of repair, with continuing growth, there is a thinning of airway walls, but a failure of alveolar development. Recognition of these two pathologically different patterns is important for further studies of the pathogenesis of bronchopulmonary dysplasia.

Comparison of treated and untreated pregnancies in a mother with phenylketonuria.

We have studied a family in which the mother has PKU and has had six pregnancies, three untreated and three treated with a low-phenylalanine diet begun during the first or second trimester. The three nonphenylketonuric offspring from two of the untreated pregnancies are mentally retarded and microcephalic. The two nonphenylketonuric offspring from two of the treated pregnancies are also microcephalic and have low or borderline low intelligence. One untreated pregnancy resulted in spontaneous abortion. One treated pregnancy resulted in stillbirth at seven months. It is not certain that dietary treatment of maternal PKU during pregnancy, when begun after conception, is sufficient to protect the fetus. This therapy may have to be ongoing at the time of conception for optimal fetal protection.

Norepinephrine-induced acute renal failure: a reversible ischemic model of acute renal failure.

The present study has defined conditions whereby a reversible form of ischemia-induced ARF can be produced in the dog. Unlike previous studies [9-11] which examined the acute phase of NE-induced ARF, this study demonstrates the feasibility of using the model for the longitudinal study of ARF. Such a model may be useful in studying the pathologic and physiologic changes which occur during different phases of ARF. Perhaps most important, this model should also provide a setting in which treatment measures, either prophylactic or therapeutic for ARF, can be examined.