Genotyping the Prop-1 mutation in Ames dwarf mice.

The Ames dwarf mouse phenotype is based on a homozygous single gene mutation in the Prop-1 gene that markedly extends life span. Since its discovery, interest in breeding these mice as a model to study retardation of aging has increased dramatically. However, the homozygous Prop-1 mutants are infertile, which necessitates breeding heterozygotes. Heterozygotes cannot be distinguished from the wildtype, while the homozygote dwarf phenotype only becomes apparent after about 3 weeks. Hence, there is a need for a simple test to genotype individual animals at an early stage for the absence or presence of one or two copies of the Prop-1 mutant gene. Here we present a Prop-1 genotyping protocol, based on a PCR reaction followed by a PflMI digestion.

Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine.

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G. C to A.T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G.C to T.A, G.C to C.G, and A.T to C.G transversions and G.C to A.T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G.C to A.T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.

Background mutations and polymorphisms in lacZ-plasmid transgenic mice.

Transgenic animal models harboring chromosomally integrated shuttle vectors with bacterial reporter genes are now widely used to measure in vivo mutant frequencies. The lacZ-plasmid transgenic mouse model has a unique sensitivity to large rearrangements compared to systems using bacteriophage lambda vectors, which mainly detect point mutations and small deletions or insertions. In this study, the background mutant frequencies and spectra in the lacZ-plasmid transgenic mouse model were investigated. While the majority of the recovered lacZ-mutants appeared to have originated in the mouse, a subset of mutants are likely to represent artifacts, and occur with a frequency of about 1.3 x 10(-5), irrespective of the total mutant frequency. Galactose-insensitive host cells, due to galE back mutations or galK or galT forward mutations, grow through the positive selection system and cause a small subset of the background. When using HindIII to excise the plasmids from genomic DNA, the largest contribution to the background, (1.1 +/- 0.3) x 10(-5), appeared to be caused by star activity, i.e., cleavage at nucleotide sequences other than the HindIII restriction enzyme recognition sequence, during the recovery procedure. Finally, a total of 10 polymorphic sites in different copies of the lacZ-plasmid cluster in founder line 60 were discovered.

Transgenic mice carrying the dominant rhodopsin mutation P347S: evidence for defective vectorial transport of rhodopsin to the outer segments.

To explore the pathogenic mechanism of dominant mutations affecting the carboxyl terminus of rhodopsin that cause retinitis pigmentosa, we generated five lines of transgenic mice carrying the proline-347 to serine (P347S) mutation. The severity of photoreceptor degeneration correlated with the levels of transgene expression in these lines. Visual function as measured by the electroretinogram was approximately normal at an early age when there was little histologic evidence of photoreceptor degeneration, but it deteriorated as photoreceptors degenerated. Immunocytochemical staining showed the mutant rhodopsin predominantly in the outer segments prior to histologically evident degeneration, a finding supported by quantitation of signal intensities in different regions of the photoreceptor cells by confocal microscopy. A distinct histopathologic abnormality was the accumulation of submicrometer-sized vesicles extracellularly near the junction between inner and outer segments. The extracellular vesicles were bound by a single membrane that apparently contained rhodopsin as revealed by ultrastructural immunocytochemical staining with anti-rhodopsin antibodies. The outer segments, although shortened, contained well-packed discs. Proliferation of the endoplasmic reticulum as reported in Drosophila expressing dominant rhodopsin mutations was not observed. The accumulation of rhodopsinladen vesicles likely represents aberrant transport of rhodopsin from the inner segments to the nascent disc membranes of the outer segments. It is possible that photoreceptor degeneration occurs because of a failure to renew outer segments at a normal rate, thereby leading to a progressive shortening of outer segments, or because of the loss of cellular contents to the extracellular space, or because of both.

Relationship between lactate dehydrogenase and myeloperoxidase levels in human gingival crevicular fluid and clinical and microbial measurements.

The present study was designed to determine, in a cross-sectional study, whether there was any relationship between levels of lactate dehydrogenase (LDH) and myeloperoxidase (MPO) in gingival crevicular fluid (GCF) and clinical periodontal status or microbial parameters. Another objective was to determine, in a longitudinal study, the effect of a single session of root planning on GCF levels of LDH and MPO and the relation to changes in clinical and microbial measurements. 15 and 12 test subjects with moderate to severe periodontal disease were seen in the cross-sectional and longitudinal study, respectively. 1 healthy and 2 diseased sites were evaluated in each subject. Higher LDH and MPO levels in GCF were closely associated with higher clinical and microbial signs of periodontal disease. Root planing was effective in reducing these enzymes in GCF, with an accompanying decrease in clinical and microbial signs associated with disease. The return of LDH to baseline levels at 3 months after instrumentation, without a corresponding return of clinical signs of disease, may serve as a marker for subclinical periodontal pathology.