Molecular cloning of thentrA gene of the broad host-rangeRhizobium sp. NGR234, and phenotypes of a site-directed mutant

Summary The clonedntrA (rpoN) gene andntrA mutants ofRhizobium meliloti were used to isolate the homologous gene from the broad-host rangeRhizobium sp. NGR234 by hybridization and interspecies complementation. The NGR234 locus was analyzed by deletion and insertional mutagenesis. A site-directedntrA mutant, NGR234rn1, was made with an interposon, GmI, and its phenotype was examined ex planta and in symbiosis. NGR234rn1 formed Fix⁻ nodules on six genera tested from among its legume hosts, including both indeterminate and determinate nodule-type plants. Formation of nodules onMacroptilium was delayed, and expression of anR. meliloti nodABC-lacZ fusion was reduced by the mutant allele.     

Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts.

A chimeric gene consisting of the coding sequence for chloroplastic Fe superoxide dismutase (FeSOD) from Arabidopsis thaliana, coupled to the chloroplast targeting sequence from the pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, was expressed in Nicotiana tabacum cv Petit Havana SR1. Expression of the transgenic FeSOD protected both the plasmalemma and photosystem II against superoxide generated during illumination of leaf discs impregnated with methyl viologen. By contrast, overproduction of a mitochondrial MnSOD from Nicotiana plumbaginifolia in the chloroplasts of cv SR1 protected only the plasmalemma, but not photosystem II, against methyl viologen (L. Slooten, K. Capiau, W. Van Camp, M. Van Montagu, C. Sybesma, D. Inzé [1995] Plant Physiol 107: 737-750). The difference in effectiveness correlates with different membrane affinities of the transgenic FeSOD and MnSOD. Overproduction of FeSOD does not confer tolerance to H2O2, singlet oxygen, chilling-induced photoinhibition in leaf disc assays, or to salt stress at the whole plant level. In nontransgenic plants, salt stress led to a 2- to 3-fold increase in activity, on a protein basis, of FeSOD, cytosolic and chloroplastic Cu/ZnSOD, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In FeSOD-overproducing plants under salt stress, the induction of cytosolic and chloroplastic Cu/ZnSOD was suppressed, whereas induction of a water-soluble chloroplastic ascorbate peroxidase isozyme was promoted.     

Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.     

Evaluation of Mitotic Activity Index in Breast Cancer Using Whole Slide Digital Images

Introduction

Mitotic Activity Index (MAI) is an important independent prognostic factor and an integral part of the breast cancer grading system. Thus, correct estimation of this prognostically relevant feature is essential for guiding treatment decision and assessing patient prognosis.The aim of this study was to validate the use of high resolution Whole Slide Images (WSI) in estimating MAI in breast cancer specimens.

Methods

MAI was evaluated in 100 consecutive breast cancer specimens by three observers on two occasions, microscopically and on WSI with a wash out period of 4 months. MAI was also translated to mitotic scores as in grading. Inter- and intra-observer agreement between microscopic and digital MAI counts and scores was measured.

Results

Almost perfect inter-observer agreements were obtained from counting MAI using a conventional microscope (intra-class correlation coefficient (ICCC) 0.879) as well as on WSI (ICCC 0.924). K coefficients reflected good inter-observer agreements among observers'' microscopic mitotic scores (average kappa 0.642). Comparable results were also observed among digital mitotic scores (average kappa 0.635). There was strong to perfect intra-observer agreements between MAI counts and mitotic scores for the two diagnostic modalities (ICCC 0.716–0.863, kappa 0.506–0.617). There were no significant differences in mitotic scores using both diagnostic modalities.

Conclusion

Scoring mitoses using WSI in breast cancer seems to be just as reliable and reproducible as when using a microscope. Further development of software and image quality will definitely encourage the use of WSI in routine pathology practice.     

Liposomes as sustained release system for human interferon-gamma: biopharmaceutical aspects

Interferon-gamma (IFNgamma) has proven to be a promising adjuvant in vaccines against cancer and infectious diseases. However, due to its rapid biodegradation and clearance, its efficacy is severely reduced. Liposomal association might prolong the residence time of IFNgamma, but no efforts have been made to optimize the biopharmaceutical characteristics of liposomal IFNgamma for its application in therapy or as vaccine immunoadjuvant. In the present study, various liposomal formulations of recombinant human IFNgamma (hIFNgamma), differing in lipid composition, were prepared via the film hydration method and characterized in vitro regarding association efficiency and bioactivity, and in vivo regarding cytokine release kinetics after subcutaneous (s.c.) administration into mice. Human IFNgamma can be formulated in large, multilamellar liposomes with high association efficiency (>80%) and preservation of bioactivity. A critical parameter is the inclusion of negatively charged phospholipids to obtain a high liposome association efficiency, which is dominated by electrostatic interactions. The fraction of externally adsorbed protein compared to the total associated protein can be minimized from 74+/-9% to 8+/-3% by increasing the ionic strength of the dispersion medium. After injection of free (125)I-hIFNgamma, the radiolabel was detectable up to 48 h at the injection site. Liposomal encapsulation of (125)I-hIFNgamma increased the local area under the curve 4-fold, and the presence of the radiolabeled hIFNgamma at the injection site was prolonged to 7 days. The release kinetics and overall residence time of the cytokine at the s.c. administration site was influenced by depletion of the externally adsorbed IFNgamma, reducing the initial burst release. Increasing the rigidity of the liposome bilayer also resulted in a more pronounced reduction of the burst release and a 19-fold increase in the residence time of the protein at the s.c. administration site, compared to the free cytokine. As adjuvanticity of liposomal IFNgamma may strongly depend on the release kinetics of cytokines in vivo, the findings in this paper may contribute to a rational design of liposomal-cytokine adjuvants in vaccines against cancer and infectious diseases.     

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells

The Bcl-2 family of proteins comprises both cell death inhibiting and cell death promoting members, generally believed to be cytoplasmic and predominantly membrane-associated. Like Bcl-2, many Bcl-2-related proteins contain a C-terminal membrane insertion domain and much research is aimed at evaluating the functional role of their localization to the outer membranes of mitochondria, the endoplasmic reticulum, and perinuclear membranes. However, confocal fluorescence microscopy of human breast cancer cells and rat colon cancer cells immunostained with commercial antibodies raised against different epitopes of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax protein revealed that these proteins are not only present in the cellular cytoplasm, but also within interphase nuclei. This was confirmed by Western blot analysis of isolated nuclei. In human cells, certain epitopes of Bcl-2, but not of Bax, were also found to be associated with mitotic chromatin. Anti-estrogen treatment of human breast cancer cells or transfection with antisense bcl-2 led to a reduction in both cytoplasmic and nuclear Bcl-2. Transfection of human bcl-2 and bax into rat cells resulted in cytoplasmic and nuclear Bcl-2 and Bax. This data seems in line with increasing evidence that the role of the Bcl-2 family of proteins should be extended to activities inside the nuclear compartment.     

Age distribution of lactating New Zealand sea lions: Interannual and intersite variation

The age distribution of 865 lactating New Zealand sea lions (NZSLs; Phocarctos hookeri ) was investigated over 3 yr (1999–2001) at two breeding colonies, Sandy Bay and Dundas Island, New Zealand. Lactating females were aged between 3 and 26 yr with a maximum observed age of 28 yr. The mean age of lactating females was 11.1 (SE = 0.16) yr. Age distributions peaked at ages 8 and 9 with a strong skew toward younger females, likely indicative of maximum recruitment into the breeding population by this age. There were significant intersite differences in age structure and also significant interannual differences in age distributions at Sandy Bay, but not at Dundas Island. Given that the two colonies are less than 10 km apart, have some interchange, and share foraging areas, these differences are surprising. However, the colony at Dundas Island is almost four times larger than Sandy Bay and may therefore be less sensitive to demographic or environmental stochasticity. That age distributions of NZSLs vary significantly over small temporal and spatial scales has important implications for the extrapolation of data from one site or year to the population level, and hence for their management and conservation.