Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

Generation of human tumor-specific CTLs in HLA-A2.1–transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu⁺ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201⁺ HER-2/neu⁺ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu⁺ tumor cell lines (also including the original HER-2/neu⁺ primary tumor cells from which the ACEs were derived) in an HLA-A*0201–restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201⁺ HER-2/neu⁺ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/neu peptides HER-2 (9₃₆₉) and HER-2 (9₄₃₅) demonstrating the immunodominance of these epitopes. HER-2 peptide–specific CTLs generated in the HLA-A*0201–transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu⁺ human tumor cell lines in an HLA-A*0201–restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide–specific CTLs (i.e., HER-2 [9₃₆₉] or HER-2 [9₄₃₅]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu⁺ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu⁺ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu⁺ malignancies.This work was supported by grants from the Regional Operational Program Attika (No. 20, MIS code 59605GR) to M.P., and from the GSRT Program (No. PENED 01ED55) to C.N.B.     

Ii-Key/HER-2/<Emphasis Type="Italic">neu</Emphasis> MHC class-II antigenic epitope vaccine peptide for breast cancer

Purpose Cytotoxic T lymphocytes (CTL)- and T-helper cell-specific, and major histocompatibility complex (MHC) class-I and class-II peptides, respectively, of the HER-2/neu protein, induce immune responses in patients. A major challenge in developing cancer peptide vaccines is breaking tolerance to tumor-associated antigens which are functionally self-proteins. An adequate CD4+ T-helper response is required for effective and lasting responses.Methods Stimulating anti-cancer CD4+ T cell responses by MHC class-II epitope peptides has been limited by their weak potency, at least compared with tight-binding MHC class-I epitope peptides. Previously, a potent T-cell response to a MHC class-II epitope was engineered by coupling the N-terminus of the pigeon cytochrome C [PGCC(95–104)] MHC class-II epitope to the C-terminus of an immunoregulatory segment of the Ii protein (hIi77–81, the Ii-Key peptide) through a polymethylene spacer.Results In vitro presentation of the MHC class-II epitope to a T hybridoma was enhanced greatly (>250 times). Now, an Ii-Key/HER-2/neu (777–789) MHC class-II epitope hybrid peptide stimulated lymphocytes from both a healthy donor and a patient with metastatic breast carcinoma. The in vitro primary stimulation with the hybrid peptide strongly activated IFN- release, whereas the epitope-only peptide was weakly active. In fact, the hybrid stimulated IFN- release as well as the wild-type peptide when augmented with IL-12; however, the hybrid was comparable to free peptide in stimulating IL-4 release. This pattern is consistent with preferential activation along a non-tolerogenic Th1 pathway.Conclusion Such Ii-Key/MHC class-II epitope hybrid peptides have both diagnostic and therapeutic applications.     

Resistance ofHER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared toHER2/neu-nonexpressing lines.HER2/neu ⁺ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in cold target inhibition experiments.HER2/neu ⁺ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and ₁ integrins was comparable to that ofHER2/neu ^– targets. In contrast, theHER2/neu ⁺ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis ofHER2/neu ^– carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression onHER2/neu ⁺ cells contributes to their resistance. The modest binding and lysis ofHER2/neu ⁺ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 onHER2/neu ⁺ targets. The following also supported deficiencies in post-binding events whenHER2/neu ⁺ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin,HER2/neu ⁺ cell lines were still resistant to LAK cytolysis, and (b)HER2/neu ⁺ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance ofHER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.Supported by research funds of the Veteran's Administration, the California Institute for Cancer Research and Jonsson Cancer Center core grant CA 16042 funded by NIH     

Effects of <Emphasis Type="Italic">in vitro</Emphasis>rooting environments and irradiance on growth and photosynthesis of strawberry plantlets during acclimatization

Strawberry (Fragaria ananassaDuch. cv. Fengxiang) plantlets were cultured under two in vitroenvironments for rooting, and then acclimatized under two ex vitroirradiance conditions. At the end of rooting stage plant height, fresh weight and specific leaf area of T1-plants grown under high sucrose concentration (3 sucrose), low photosynthetic photon flux density (30 mol m^–2 s^–1) and normal CO₂ concentration (350–400 l l^–1) were significantly higher than those of T2-plantlets grown under low sucrose concentration (0.5), high photosynthetic photon flux density (90 mol m^–2 s^–1) and elevated CO₂ concentration (700–800 l l^–1). But T2-plantlets had higher net photosynthetic rate (Pn), effective photochemical quantum yield of PSII (_PSII), effective photosynthetic electron transport rate (ETR), photochemical quenching (q_P) and ratio of chlorophyll fluorescence yield decrease (Rfd). After transfer, higher irradiance obviously promoted the growth of plantlets and was beneficial for the development of photosynthetic functions during acclimatization. T2-plantlets had higher fresh weight, leaf area, _PSII and ETR under higher ex vitroirradiance condition.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 g ml^–1 CdCl₂ in the presence or absence of 0.1–1000 ng ml^–1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17-estradiol (E₂) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 g ml^–1 CdCl₂ suppressed P accumulation to 65 and 10%, respectively; accumulation of E₂ (at 5 g ml^–1 CdCl₂) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 g ml^–1 CdCl₂ was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 g ml^–1 CdCl₂, whereas basal E₂ production of granulosa cells was markedly diminished. It is concluded that CdCl₂ suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.     

A CD80-transfected human breast cancer cell variant induces HER-2/neu–specific T cells in HLA-A*02–matched situations in vitro as well as in vivo

Adjuvant treatment is still only working in a small percentage of breast cancer patients. Therefore, new strategies need to be developed. Immunotherapies are a very promising approach because they could successfully attack tumor cells in the stage of dormancy. To assess the feasibility of using an allogeneic approach for vaccination of breast cancer patients, we selected a CD80-transfected breast cancer cell line based on its immunogenic properties. Using CD80⁺ KS breast cancer cells and human leukocyte antigen (HLA)-A*02–matched peripheral blood mononuclear cells (PBMCs) of breast cancer patients in allogeneic mixed lymphocyte–tumor cell cultures (MLTCs), it was possible to isolate HLA-A*02–restricted cytotoxic T cells (CTLs). Furthermore, a genetically modified KS variant expressing influenza A matrix protein serving as a surrogate tumor-associated antigen (TAA) was able to stimulate flu peptide-specific T cells alongside the induction of alloresponses in MLTCs. KS breast cancer cells were demonstrated to express already known TAAs such as CEA, MUC-1, MAGE-1, MAGE-2, and MAGE-3. To further improve antigenicity, HER-2/neu was added to this panel as a marker antigen known to elicit HLA-A*02–restricted CTLs in patients with breast cancer. Thus, the antigen-processing and antigen-presentation capacity of KS cells was further demonstrated by the stimulation of HER-2/neu–specific CD8⁺ T cells in PBMCs of breast cancer patients in vitro. These results gave a good rationale for a phase I/II trial, where the CD80⁺ HER-2/neu–overexpressing KS variant is actually used as a cellular vaccine in patients with metastatic breast cancer. As a proof of principle, we present data from two patients where a significant increase of interferon- (IFN-) release was detected when postvaccination PBMCs were stimulated by allogeneic vaccine cells as well as by HLA-A*02–restricted HER-2/neu epitopes. In whole cell vaccine trials, monitoring is particularly challenging because of strong alloresponses and limited knowledge of TAAs. In this study, a panel of HER-2/neu epitopes, together with the quantitative real time (qRT)-PCR method to analyze vaccine-induced cytokines secreted by T cells, proved to be highly sensitive and feasible to perform an immunological staging following vaccination.     

INFLUENCE DU SUPERPARASITISME SUR LA DURÉE DU DÉVELOPPEMENT LARVAIRE ET LE POIDS DU PARASITOÏDE LIXOPHAGA DIATRAEAEÉLEVÉ DANS UN HÔTE DE SUBSTITUTION GALLERIA MELLONELLA

Résumé G. mellonella infestée au dernier stade larvaire avec 1, 2, 3 ou 5 planidia/hôte (ph/H) produit 1 à 5 pupes/hôte (pu/H). La mortalité des chenilles augmente avec le nombre de pl/H. Le poids des pupes et décroît avec un nombre croissant de pu/H (18,2 à 12,9 mg pour les et 12,5 à 9,7 mg pour les ). Le développement larvaire dure 8,7 j. chez les et 8,3 chez les ; il est peu affecté par le superparasitisme. Avec 1, 2, 3 et 5 pl/H nous obtenons 0,84–1,61–2,17 et 3,43 pu/H et 0,81–1,48–2,10 et 3,11 imagos/H. L'optimum est de 3 pl/H ou 1 à 2=pl/H pour obtenir des parasitoïdes plus lourds.

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Summary The influence of superparasitism on larval and pupal development is investigated. Last-larval instar G. mellonella (200±5 mg) were infected with 1, 2, 3 or 5 planidia/host (pl/H) producing 1 to 5 pupae per host (pu/H). Host mortality (8.6–8.3–14.3 and 22.2%) increased with the number of planidia. The planidia may transmit a bacteriosis. Pupal weight ( and ) decreased as number of pu/H increased. pupae were heavier than ones: 12.9 to 18.2 mg for against 9.7 to 12.5 mg for . Larval development lasted slightly longer for (8.7 d.) than for (8.3 d.), and its duration was little affected by superparasitism. 1, 2, 3 or 5 pl/H yielded 0.84–1.61–2.17 and 3.43 pu/H and 0.81–1.48–2.10 and 3.11 adults/H. An optimum was obtained with 3 planidia of L. diatraeae on G. mellonella or 1 to 2 to obtain heavier parasitoids.

     

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828–836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu ⁺ tumor cell lines. HER-2/neu(828-836), [HER-2(9₈₂₈)], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9₈₂₈) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8⁺ T-lymphocytes specifically recognizing HER-2(9₈₂₈) in 8 out of 20 HLA-A*0201⁺ HER-2/neu ⁺ breast cancer patients. Moreover, HER-2(9₈₂₈)-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9₈₂₈) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9₈₂₈) as a promising candidate for peptide-based cancer vaccines.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

Whilst reliable estimates of chiasma frequencies can usually not be obtained, the probability (b) of a chromosome arm to be bound by at least one chiasma can often be determined. In the absence of interference this probability equals (1–e ^–2), where 2 is the average chiasma frequency of the chromosome arm and the average crossover frequency or map length. In the presence of interference is shown to retain its genetic meaning as an additive metric that may describe the chromosome arm or other distinctive chromosome segment in terms of genetic recombination. It is a form of potential map length, comparable to, but numerically different from the regular map length. It is termed provisionally crossing-over potential.A chromosome with armsm andn with crossing-over potentials and will form ring bivalents with a frequency (1–e ^–2).(1–e ^–2); open bivalents with a frequency (1–e ^–2).e ^–2+(1–e ^–2).e ^–2; univalent pairs with a frequencye ^–2.e ^–2. Estimates of these frequencies yield equations from which and may be solved. In rye (Secale cereale) their ratio (q) is approximately two and differs from the mitotic arm length ratio of 1.4, indicating localization of chiasmata in the long arms.Graphs are given to show how, with constantq, the relation between the probabilitiesb _m andb _n of the two arms being bound changes with changing averageb.Data are presented on chiasma frequencies in M I, and compared with the frequencies expected in the absence of interference to give an impression of the degree of interference. Apparent fusion of chiasmata simulates interference.     

Photosynthetic light-use by three bromeliads originating from shaded sites (<Emphasis Type="Italic">Ananas ananassoides,Ananas comosus</Emphasis> cv. Panare) and exposed sites (<Emphasis Type="Italic">Pitcairnia pruinosa</Emphasis>) in the medium Orinoco basin,Venezuela

Three Bromeliaceae species of the medium Orinoco basin, Venezuela, were compared in their light-use characteristics. The bromeliads studied were two species of pineapple, i.e. the wild species Ananas ananassoides originating from the floor of covered moist forest, and the primitive cultivar Panare of Ananas comosus mostly cultivated in semi-shaded palm swamps, and Pitcairnia pruinosa, a species abundant in highly sun exposed sites on rock outcrops. Ananas species are Crassulacean acid metabolism (CAM) plants, P. pruinosa is C₃ plant. Plants were grown at low daily irradiance (LL = 1.3 mol m^–2 d^–1 corresponding to an incident irradiance of 30 mol m^–2 s^–1) and at high irradiance (HL = 14.7 mol m^–2 d^–1 or 340 mol m^–2 s^–1), and CO₂ and H₂O-vapour gas exchange and photochemical (q_P) and non-photochemical quenching (q_NP) of chlorophyll a fluorescence of photosystem 2 (PS2) were measured after transfer to LL, medium irradiance (ML = 4.1 mol m^–2 d^–1 or 95 mol m^–2 s^–1) and HL. All plants showed flexible light-use, and q_P was kept high under all conditions. LL-grown plants of Ananas showed particularly high rates of CAM-photosynthesis when transferred to HL and were not photoinhibited.     

Characterization of NO adducts of the diiron center in protein R2 of<Emphasis Type="Italic"> Escherichia coli</Emphasis> ribonucleotide reductase and site-directed variants; implications for the O<Subscript>2 </Subscript>activation mechanism*

The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O₂ to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [Fe^III-Fe^IV] state called compound X, but in related diiron proteins such as methane monooxygenase, ⁹-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate (-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O₂ activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}⁷]₂ center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}⁷ dimer where both Fe–N–O units share a common vibrational signature. In the wt protein, (Fe–NO), (Fe–N–O), and (N–O) bands are observed at 445, 434 and 1742 cm^–1, respectively, while in the variant proteins the (Fe–NO) and (Fe–N–O) bands are observed ~10 cm^–1 higher and the (N–O) ~10 cm^–1 lower at 1735 cm^–1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}⁷]₂ species with two identical Fe–N–O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O₂ activation process in R2-wt.     

Age-Related Changes in Oxygen Consumption in the Edible Mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> from the White Sea

We determined the rate of oxygen consumption in the White Sea edible mussel Mytilus edulis of different ages. The mass-specific rate of oxygen consumption proved to decrease with mussel age according to the equation: = 40.1/(1 – e ^–0.194t ), where is mass-specific rate of oxygen consumption and t is age. Allometric coefficients of the oxygen consumption rate–soft tissue weight relationship were also determined.     

Expression of Autophagy-Related Proteins According to Androgen Receptor and HER-2 Status in Estrogen Receptor-Negative Breast Cancer

Purpose

The purpose of this study was to investigate the expression of autophagy-related proteins in relation to androgen receptor (AR) status in estrogen receptor (ER)-negative breast cancers.

Methods

We extracted 334 ER-negative breast cancer samples to construct tissue microarrays (TMAs), which were immunohistochemically stained for autophagy-related proteins (beclin-1, LC3A, LC3B, p62) and for AR and HER-2.

Results

There were 127 AR-positive cases and 207 AR-negative cases, and 140 HER-2-positive cases and 194 HER-2 negative cases. The AR-negative group was associated with tumoral LC3A expression (P<0.001), while the AR-positive group was associated with tumoral BNIP3 expression (P<0.001). Tumoral LC3A was most highly expressed in the AR-negative and HER-2 negative group, while stromal LC3A showed the highest expression in the AR-negative and HER-2-positive group. Tumoral BNIP3 and stromal BNIP3 were highest in the AR-positive and HER-2-negative group. In the AR-positive and HER-2-negative group, stromal p62 positivity was an independent factor that was statistically significant in its association with shorter disease-free survival (DFS) (Hazard ratio: 10.21, 95% CI: 1.130–92.31, P = 0.039). Shorter DFS was associated with tumoral LC3A positivity (Hazard ratio: 10.28, 95% CI: 2.068–51.19, P = 0.004) in the AR-negative and HER-2-positive group.

Conclusion

In ER-negative breast cancers, AR status was associated with expression of different types of autophagy-related proteins. Tumoral LC3A was most highly expressed in AR-negative breast cancers, while tumor BNIP3 was highest in AR-positive breast cancers.     

Stable carbon isotope ratios for the gray whale (<Emphasis Type="Italic">Eschrichtius robustus</Emphasis>) in the breeding grounds of Baja California Sur,Mexico

This work investigated the feeding ecology and behaviour of gray whales in Bahía Magdalena. Underwater observations of bottom feeding were made (n=4). Skin biopsies of the gray whale had a carbon isotope value of –16.5 ± 0.1 (range from –16.4 to –16.7, n=7). Prey in Bahía Magdalena had a carbon isotope value of –18.4. Dietary enrichment from prey in Bahía Magdalena would correspond to 2 ± 0.1, whereas previously published results for prey in Alaska would result in an enrichment of 3, which suggests that whales were more likely feeding on prey from Bahía Magdalena. Carbon isotopic oscillation along the baleen plate of a stranded 1-year-old whale showed a variation in diet during the year, which suggests continual feeding during this time and corresponding to dietary sample measurements from Bahía Magdalena in winter and Alaska in summer.     

Effects of CO<Subscript>2</Subscript> concentration on acclimatization and physiological responses of two cultivars of carob tree

This study reports survival and physiological responses of micropropagated Ceratonia siliqua L. cvs. Galhosa and Mulata plants during ex vitro acclimatization under ambient (AC; 330 mol mol^–1) or elevated (EC; 810 mol mol^–1) CO₂ concentration and a photosynthetic photon flux density of 125 mol m^–2 s^–1. CO₂ enrichment during acclimatization did not improve survival rate that was around 80 % for both treatments. Eight weeks after ex vitro transplantation, photosynthetic capacity and apparent quantum yield in acclimatized leaves were higher in comparison with those in in vitro-grown leaves, without any significant difference between CO₂ treatments. Chlorophyll content increased after acclimatization. However, EC led to a decrease in the total amount of chlorophyll in new leaves of both cultivars, compared to those grown at AC. Soluble sugars and starch contents were not markedly affected by growth EC, although starch had significantly increased after transfer to ex vitro conditions. EC induced an increase in the stem elongation and in the effective life of leaves, and a decrease in the number of new leaves.     

Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties

Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamadaet al., 1979).By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP^2– and MgADP^–) and (b) the uncomplexed nucleotide substrates (ADP^3– and AMP^2–) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15–25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of AMP.The syntheses are described as a set of peptides corresponding to residues 31–45, 20–45, 5–45, and 1–45, and a set of peptides corresponding to residues 178–192, 178–194, and 172–194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: MgATP/ATP and MgADP/ADP are quantitatively presented in terms of their intrinsic dissociation constants (K_d) and values ofN (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1–44) obtained from rabbit muscle myokinase (Kubyet al., 1984) and with the native enzyme (Hamadaet al., 1979). In addition, the values ofN andK_d are given for the second set of synthetic peptides to the fluorescent ligands AMP and ADP as well as for the peptide fragments MT-XII(172–194) and CB-VI(126–194) (Kuby et al., 1984) and, in turn, compared with the native enzyme.A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., theK _i for AMP and the value of obtained for the native enzyme) (Hamada and Kuby, 1978), and theK'_d measured for Cr³⁺ and the synthetic peptide I_1–45 (Fryet al., 1985b).Paper XVII of this series is Kubyet al. (1983).     

Changes in the Photosynthetic Apparatus of Broad Bean Leaves as Dependent on the Content of Heavy Metals in the Growth Medium

The content of chlorophyll, the rate of O₂ evolution, the slow phase of fluorescence induction, and photoinduced changes in the intensity of electron paramagnetic resonance (EPR) signal I from the reaction center of photosystem I (P⁺700) were studied in leaves of Vicia faba L. grown in 10^–7–10^–3 M aqueous solutions of CdCl₂, SnCl₂, CuCl₂, and MgCl₂. At low concentrations of heavy metal (Cd, Sn, and Cu) chlorides, the content of chlorophyll per fresh weight decreased. However, the rate of O₂ evolution calculated per chlorophyll basis, O₂/(t chlorophyll), increased. High concentrations of heavy metals significantly suppressed plant development and inhibited photosynthetic O₂ evolution. In contrast, plant treatment with MgCl₂ (10^–5–10^–3 M) resulted in an increase in the content of chlorophyll and a stimulation of leaf photosynthetic activity. A positive correlation between the F _M/F _T ratio and O₂/(t chlorophyll) (r = 0.89, P > 0.999) was found. We observed a negative correlation between the values of P/P ₀ of EPR signal I (white/far-red light) and O₂/(tchlorophyll) (r = –0.89, P > 0.999). The data obtained indicate nonspecific and nonmonotone changes in the photosynthetic apparatus of plants treated with heavy metals: low concentrations of heavy metals (10^–7–10^–6 M) stimulated photosynthetic activity, whereas high concentrations (10^–4–10^–3 M) suppressed it.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.