Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Translation inhibitors induce cell death by multiple mechanisms and Mcl-1 reduction is only a minor contributor

There is significant interest in treating cancers by blocking protein synthesis, to which hematological malignancies seem particularly sensitive. The translation elongation inhibitor homoharringtonine (Omacetaxine mepesuccinate) is undergoing clinical trials for chronic myeloid leukemia, whereas the translation initiation inhibitor silvestrol has shown promise in mouse models of cancer. Precisely how these compounds induce cell death is unclear, but reduction in Mcl-1, a labile pro-survival Bcl-2 family member, has been proposed to constitute the critical event. Moreover, the contribution of translation inhibitors to neutropenia and lymphopenia has not been precisely defined. Herein, we demonstrate that primary B cells and neutrophils are highly sensitive to translation inhibitors, which trigger the Bax/Bak-mediated apoptotic pathway. However, contrary to expectations, reduction of Mcl-1 did not significantly enhance cytotoxicity of these compounds, suggesting that it does not have a principal role and cautions that strong correlations do not always signify causality. On the other hand, the killing of T lymphocytes was less dependent on Bax and Bak, indicating that translation inhibitors can also induce cell death via alternative mechanisms. Indeed, loss of clonogenic survival proved to be independent of the Bax/Bak-mediated apoptosis altogether. Our findings warn of potential toxicity as these translation inhibitors are cytotoxic to many differentiated non-cycling cells.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Inhibitory effect of human syncytiotrophoblast plasma membrane vesicles on Jurkat cells activated by phorbol ester and calcium ionophore.

The effects of syncytiotrophoblast plasma membrane vesicles (STPM) on stimulated Jurkat leukemic T cells have been investigated. STPM inhibited IL-2 production and the expression of protein P55 of the IL-2 receptor (IL-2R P55), when Jurkat cells were stimulated by a combination of calcium ionophore A23187 (CaI) + phorbol 12-myristate 13-acetate (PMA). STPM also inhibited IL-2R P55 when cells were stimulated by PMA alone, a situation in which IL-2 production is negligible. On the other hand, STPM had no effect on the sustained mobilization of intracellular Ca2+ induced by CaI nor on the PKC-dependent CD3 down regulation induced by PMA. Finally STPM had no effect on intracellular cAMP levels. These results show that (i) the inhibitory effect of STPM on IL-2R P55 expression is independent of the inhibition of IL-2 production, and (ii) the inhibitory effects of STPM are at least partially independent of phosphatidylinositol 4,5-bisphosphate hydrolysis. They suggest that STPM affect a signaling pathway activated by PMA but possibly PKC independent.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

Isolation and characterization of syncytiotrophoblast plasma membrane from human placenta

Human full-term syncytiotrophoblast plasma membranes isolated by mechanical procedures (sieving and ultrasonic disintegration), purified by phase centrifugation, form a single band of 1.052 +/- 0.002 g/ml density in percoll gradient. The purity of the preparation was assessed by electron microscopy, enzyme analysis and beta 2-microglobulin determination.     

Cyclic amp-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus: Expression of transformation-associated markers

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by α-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.     

Trophoblast cell line resistance to NK lysis mainly involves an HLA class I-independent mechanism

The lack of classical HLA molecules on trophoblast prevents allorecognition by maternal T lymphocytes, but poses the problem of susceptibility to NK lysis. Expression of the nonclassical class I molecule, HLA-G, on cytotrophoblast may provide the protective effect. However, the class I-negative syncytiotrophoblast escapes NK lysis by maternal PBL. In addition, while HLA-G-expressing transfectants of LCL.721.221 cells are protected from lymphokine-activated killer lysis, extravillous cytotrophoblast cells and HLA-G-expressing choriocarcinoma cells (CC) are not. The aim of this work was therefore to clarify the role of HLA class I expression on trophoblast cell resistance to NK lysis and on their susceptibility to lymphokine-activated killer lysis. Our results showed that both JAR (HLA class I-negative) and JEG-3 (HLA-G- and HLA-Cw4-positive) cells were resistant to NK lysis by PBL and were equally lysed by IL-2-stimulated PBL isolated from a given donor. In agreement, down-regulating HLA class I expression on JEG-3 cells by acid treatment, masking these molecules or the putative HLA-G (or HLA-E) receptor CD94/NKG2 and the CD158a/p58.1 NKR with mAbs, and inducing self class I molecule expression on JAR cells did not affect NK or LAK lysis of CC. These results demonstrate that the resistance of CC to NK lysis mainly involves an HLA class I-independent mechanism(s). In addition, we show that the expression of a classical class I target molecule (HLA-B7) on JAR cells is insufficient to induce lysis by allospecific polyclonal CTL.