Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin-fixed lymphoid tissues

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin-embedded, formalin-fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin-fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S-phase fraction was higher in paraffin-embedded tissues than in unfixed cells. This difference could be attributed to 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI), a DNA-binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin-embedded tissues generally showed a greater S-fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S-fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.     

Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5.     

Objectives and Program of the A.M.A. Committee on Nursing

The continued achievement of high standards of patient care in the preventive, curative, and restorative aspects of illness depends upon a harmonious, collaborative relationship between medicine and nursing. In an effort to protect and foster an enduring alliance of understanding and cooperation between these 2 major health professions, the Committee on Nursing has instituted a continuing program of liaison, communication, education, and research. The Committee has authorized publication of the following report on its objectives and program.     

The effect of externally applied electrostatic fields,microwave radiation and electric currents on plants and other organisms,with special reference to weed control

A wide-ranging review is presented of the effects of various forms of externally applied electrical energy upon plants and other organisms. Although investigations involving both small and large amounts of energy directed at the targets are considered, a particular emphasis of this review is the feasibility of each type of electrical stimulation for weed control. Electrostatic fields ranging from 100 V m^?1 to 800 kV m^?1 have been applied to plants under laboratory conditions and in field trials since the 1880’s. Some beneficial effects have been reported (e.g. increase in yield from both cereal and vegetable crops), but the results have been erratic and the electrical conditions leading to definite benefits on a large scale could not be confidently predicted from early studies. High electric fields are reported to damage plants if currents greater than 10^?6 A are induced to flow through leaves causing corona discharges from the tips. The nature of the damage and the effects on metabolic processes are discussed. The results from experiments on the growth of plants in which the density and charge of air ions have been varied are also reviewed. The effects of microwave radiation (mostly 2450 MHz) upon seeds, plants and other organisms in soil are discussed. These effects depend upon the power density of the radiation and the electrical properties of the targets. Factors such as size of seeds and plants, shape and moisture content are important, as are the properties of the soil irradiated (notably water content). Although microwaves can be effective in killing plants and also seeds that are buried several centimeters deep in soil, high power equipment is required and treatment times are long e.g. a 60 kW machine could take up to 92.6 hours per hectare. Other experiments reported show that microwave radiation can kill nematodes in the soil and that it is also very effective in killing fungi and bacteria. The potential of the various possible uses of microwave radiation in agriculture is also described. Electric currents have been caused to flow through plants by the application of electrodes to the leaves. The effects range from nil, when 50–100 V and 1 or 2 μA are used, to very striking when voltages from 5 to 15 kV are applied causing currents of several amperes to flow and resulting in the rapid destruction of the target. Small electric currents passed through soil containing plants are reported to increase their growth. The effects of small current on the growth of individual leaves are reviewed. The use of high voltage tractor-borne equipment for weed control is also considered.     

Studies on the effect of reagent and protein charges on reactivity of the β93 sulfhydryl group of human hemoglobin using selected mutations

The reactivity of the β93 sulfhydryl (SH) group of human oxyhemoglobins with the negatively charged 5,5′-dithiobis(2,2′-nitrobenzoate) and the uncharged 2,2′-dithiodipyridine was determined as a function of pH. Selected mutant hemoglobins having increased oxygen affinity and having residue substitutions altering charge near the SH group (Wood, Malmö, Yakima, Kempsey, Andrew-Minneapolis, Osler, and Chesapeake) were compared to hemoglobin (Hb) A. Although both reagents reacted with GSH at the same rate and with the same enthalpies of activation, the rates with Hb were different and the difference showed a pronounced pH dependence. The charged reagent was sensitive to charges near the SH group; a positive charge increased the rate and a negative one decreased the rate. The uncharged reagent which reacted with Hb A with activation enthalpies similar to those for GSH was insensitive to neighboring charges, but was sensitive to tertiary and quaternary structural changes. The rates obtained with the latter reagent did not correlate with oxygen affinity. The evolutionary aspects of the β93 cysteine in relation to structure and function are reviewed.