Establishing a molecular continuum in breast cancer DNA microarrays and benign breast disease.

Although the risk of breast cancer for women in the United States is approximately 1 in 9, identification of risk factors and translation of that knowledge into strategies for prevention have been inhibited by poor understanding of disease pathogenesis. A few benign breast proliferations are associated with higher risks of breast cancer, but definition of a preneoplastic morphologic continuum is lacking. If progression from a premalignant state to malignancy is accompanied by genetic changes, then identification in benign breast disease lesions (BBD) of alterations similar to those found in breast cancer should strengthen the perception of BBD as a premalignant condition. Current testing for hereditary breast cancer susceptibility presumes that only women with invasive breast or ovarian cancer are gene carriers. Therefore, neither in situ breast cancer nor atypical hyperplasias are considered clinically as evidence of a breast-ovarian syndrome, nor are these diagnoses used to predict carrier status within at-risk families. This reflects lack of evidence that breast cancer develops along a recognized morphologic continuum from precursor lesions. New mutation screening procedures such as DNA microarrays can provide sensitivity, specificity, and high throughput that circumvent limitations imposed on the scope of molecular marker analyses applied to archival resources. We have studied a BRCA1-mutant individual with loss of the wild type BRCA1 allele in benign breast proliferations. Both her benign and malignant lesions showed molecularly identical TP53 mutations, indicating that significant genetic alterations can occur in BBD and supporting the clonal evolution from BBD to malignancy.     

Are the polarization colors of Picrosirius red-stained collagen determined only by the diameter of the fibers?

Summary Polarization colors of various purified collagens were studied in fibers of similar thickness. Three different soluble collagens of type I, insoluble collagen type I, lathyritic collagen type I, two p-N-collagens type I, pepsin extract collagen type II, two soluble collagens type III, p-N-collagen type III, and soluble collagen type V were submitted to a routine histopathologic procedure of fixation, preparation of 5-m-thick sections, staining with Picrosirius red and examination under crossed polars. Polarization colors were determined for thin fibers (0.8 m or less) and thick fibers, (1.6–2.4 m). Most thin fibers of collagens and p-N-collagens showed green to yellowish-green polarization collors with no marked differences between the various samples. Thick fibers of all p-N-collagens, lathyritic and normal 0.15 M NaCl-soluble collagens showed green to greenish-yellow polarization colors, while in all other collagens, polarization colors of longer wavelengths (from yellowish-orange to red) were observed. These data suggested that fiber thickness was not the only factor involved in determining the polarization colors of Picrosirius red-stained collagens. Tightly packed and presumably, better aligned collagen molecules showed polarization colors of longer wavelengths. Thus, packing of collagen molecules and not only fiber thickness plays a role in the pattern of polarization colors of Picrosirius red-stained collagens.     

An attempt to prevent or delay denervation changes in rat muscles

A number of drugs which are known to affect lysosomes and their enzyme activities were used in an attempt to inhibit or delay the onset of denervation changes in rat muscles. The following parameters were used: the occurrence of fibrillations in electromyographs; diameters of muscle fibers; acid phosphatase activity; acetylcholinesterase activity and distribution in end plates. Differences between denervated and non-denervated limbs were evaluated and compared in the different treatment groups. The various parameters were differently affected by the different drugs. Chloroquin, thiouracil and streptomycin appeared to be more effective than other treatments in the inhibition of denervation changes.     

Effect of exercise on protein metabolism in humans as explored with stable isotopes

Exercising for 3.75 h on a treadmill at 50% VO2 max in the fed state induced an increased excretion of 71 mg nitrogen/kg over the 18 h after exercise. However, measurements of the time course of changes in 13CO2 excretion from ingested [1-13C]leucine indicated that all of this increased nitrogen production occurs during the exercise period. Because of the reduced renal clearance and slow turnover of the urea pool, urea excretion lags behind urea production. Measurements of nitrogen flux from the plateau labeling of urinary ammonia achieved by repeated oral doses of 15N-labeled glycine indicated that the nitrogen loss resulted from an increase in protein degradation and a decrease in protein synthesis. Further studies with [1-13C]leucine indicated that a 2-h treadmill exercise induced an increase in the nitrogen loss from 5.4 to 16 mg . kg-1 . h-1 measured with a primed constant infusion of [1-13C]leucine. This resulted from a fall in whole-body protein synthesis. Glucose given at the rate of 0.88 g . kg-1 . h-1 depressed the rate of whole-body protein degradation and appeared to suppress the exercise-induced increase in nitrogen excretion. When leucine oxidation rates were measured at increasing work rates, a linear relationship between percentage of VO2 max and leucine oxidation was observed up to 89% VO2 max when 54% of the flux of leucine was oxidized. These changes may involve nonmuscle as well as muscle tissue. Thus the source of the increased nitrogen losses is probably liver. In muscle, protein degradation is actually decreased judged by methylhistidine excretion, whereas in liver, protein degradation may be increased. Also the fall in whole-body protein synthesis may reflect changes in nonmuscle tissues because in running rats protein synthesis in muscle is maintained. As far as leucine metabolism is concerned, because the increase in leucine oxidation occurs when leucine and its keto acid concentration falls, exercise must specifically activate the 2-oxoacid dehydrogenase.     

Differential Staining of Acidic Tissue Components by the Improved Bi-Col Method

Technical particulars of the Bi-Col method for differentiating strongly acidic from weakly acidic compounds are described. The method can be applied to tissue sections which should preferably be fixed in Carnoy's fixative, and to strips of glass fiber paper.     

Triazinic dye ligand selection by surface plasmon resonance for recombinant lactoferricin purification

Bovine lactoferricin (Lfcin B) belongs to the antimicrobial peptide family, which is the first line of defense against pathogens in many organisms. Lfcin B has important applications due to its antiviral, antifungal, antiparasitic, anticancer/tumor and antibacterial activity.In this work, we tested five triazine dyes for Lfcin B affinity interactions using surface plasmon resonance (SPR) technology. Recombinant Lfcin B was expressed as a fusion protein with GST (Lfcin B-GST) by using the baculovirus expression vector system and the dye-Sepharose matrices were assayed for Lfcin B-GST adsorption and subsequent elution.Red HE-3B and Yellow HE-4R dyes were selected and immobilized on a Sepharose-4B matrix for further purification studies. The Yellow HE-4R-Sepharose matrix was specific for Lfcin B and allowed adsorption of Lfcin B-GST directly from the culture medium even at high salt concentration.This novel application of SPR to screen possible dye–peptide interactions could be relevant to purify other peptides or proteins by using low-cost dye-affinity chromatography.