Reproductive biology of Olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) DOP Umbria cultivars

Olive trees have a plentiful bloom but a low percentage of normal fruit set. To improve fruit set, numerous investigations have sought to identify the obstacles that prevent full production. In this work, flower development in five DOP Umbria cultivars (Leccino, Frantoio, Moraiolo, Dolce Agogia and San Felice) was studied throughout different developmental phases, from before microsporogenesis and megasporogenesis to post-anthesis, by morphological and cyto-histological observations. Dolce Agogia was the most precocious cultivar, while full flowering was simultaneous in Leccino, Frantoio, Moraiolo and San Felice. Frantoio and Leccino were also good pollen producers, having the highest percentage of pollen viability and germinability. Dolce Agogia can also be considered a good pollen producer in terms of the high quantity of released pollen, but it had the lowest levels of pollen viability and germinability and the highest percentage of aborted flowers and ovaries. Morphological and cyto-histological observations on the number of flowers per inflorescence and the number of aborted flowers and ovaries suggest that fruit set was not influenced by the number of flowers per inflorescence, but rather by the number of inflorescences, which depends on the global fruiting potential of the tree.     

Increased bone marrow interleukin-7 (IL-7)/IL-7R levels but reduced IL-7 responsiveness in HIV-positive patients lacking CD4+ gain on antiviral therapy

Background

The bone marrow (BM) cytokine milieu might substantially affect T-lymphocyte homeostasis in HIV-positive individuals. Interleukin-7 (IL-7) is a bone marrow-derived cytokine regulating T-cell homeostasis through a CD4+-driven feedback loop. CD4+ T-lymphopenia is associated with increased free IL-7 levels and reduced IL-7R expression/function, which are only partially reverted by highly active antiretroviral therapy (HAART). We investigated the BM production, peripheral expression and signaling (pStat5+ and Bcl-2+ CD4+/CD8+ T cells) of IL-7/IL-7Rα in 30 HAART-treated HIV-positive patients who did not experience CD4+ recovery (CD4+ ≤200/µl) and who had different levels of HIV viremia; these patients included 18 immunological nonresponders (INRs; HIV-RNA≤50), 12 complete failures (CFs; HIV-RNA>1000), and 23 HIV-seronegative subjects.

Methods

We studied plasma IL-7 levels, IL-7Rα+CD4+/CD8+ T-cell proportions, IL-7Rα mRNA expression in PBMCs, spontaneous IL-7 production by BM mononuclear cells (BMMCs), and IL-7 mRNA/IL-7Rα mRNA in BMMC-derived stromal cells (SCs). We also studied T-cell responsiveness to IL-7 by measuring the proportions of pStat5+ and Bcl-2+ CD4+/CD8+ T cells.

Results

Compared to HIV-seronegative controls, CFs and INRs presented elevated plasma IL-7 levels and lower IL-7Rα CD4+/CD8+ cell-surface expression and peripheral blood production, confirming the most relevant IL-7/IL-7R disruption. Interestingly, BM investigation revealed a trend of higher spontaneous IL-7 production in INRs (p = .09 vs. CFs) with a nonsignificant trend toward higher IL-7-Rα mRNA levels in BMMC-derived stromal cells. However, upon IL-7 stimulation, the proportion of pStat5+CD4+ T cells did not increase in INRs despite higher constitutive levels (p = .06); INRs also displayed lower Bcl-2+CD8+ T-cell proportions than controls (p = .04).

Conclusions

Despite severe CD4+ T-lymphopenia and a disrupted IL-7/IL-7R profile in the periphery, INRs display elevated BM IL-7/IL-7Rα expression but impaired T-cell responsiveness to IL-7, suggesting the activity of a central compensatory pathway targeted to replenish the CD4+ compartment, which is nevertheless inappropriate to compensate the dysfunctional signaling through IL-7 receptor.     

Immunoconjugate generation between the ribosome inactivating protein restrictocin and an anti-human breast carcinoma MAB

Summary Treatment of Wistar/AG rats with a single i.p. injection of 1 mg/kg of synthetic double-stranded polynucleotides, either polyadenylic-polyuridylic acid (rA_n.rU_n), or a mismatched analogue of polyinosinic-polycytidylic acid (rI_n.r(C₁₂U)_n), enhanced the cytotoxicity of natural killer (NK) cells among peripheral blood leukocytes and lung intracapillary leukocytes (LICL). The enhancement reached a peak 24 h after treatment and returned to control values after 4 days. In rats given repeated injections of double-stranded polynucleotides (2 per week), the NK cytotoxicity expressed by LICL reached more than ten times (in lytic units) the control levels between day 8, after 3 injections, and day 360, after 100 injections. No hypore-sponsiveness was observed. Moreover, NK activity was frequently and significantly higher in rats given multiple injections than in those given a single injection. In rats with experimentally induced P77 lung fibrohistiocytoma colonies, repeated injections of rI_n.r(C₁₂U)_n stimulated NK activity and reduced the number of metastatic nodules from 172 to 19. The same significant reduction (from 172 to 27) was also observed in animals given repeated injections of rA_n.rU_n. However, with two models of spontaneous metastases, significant reduction in lung metastases (M37 bronchioloalveolar carcinoma) or lack of effect (S₄T₁₉ rhabdomyosarcoma) were observed.     

Cortical-sparing laparoscopic adrenalectomy in a patient with multiple endocrine neoplasia type IIA

We describe the case of a patient affected by multiple endocrine neoplasia type IIA with a new diagnosis of an asymptomatic right pheochromocytoma. The patient underwent laparoscopic adrenalectomy with adrenal sparing. The removal of the tumor was successful with preservation of about one third of the adrenal gland. At the time of the last follow-up, the patient is well with partial hypoadrenalism without replacement therapy. The limitations to cortical-sparing adrenalectomy imposed by traditional open surgery (small tumor with peripheral location) can be reconsidered using the laparoscopic approach. Laparoscopic cortical-sparing adrenalectomy should become the gold standard for treatment of bilateral pheochromocytoma. The advantages of this technique are its efficacy and its reduced invasiveness with a low rate of complications either during the operation or in the postoperative period. Moreover, the preservation of a portion of the adrenal cortex may prevent the need for a life-long steroid replacement therapy.     

Filipin-dependent Inhibition of Cholera Toxin: Evidence for Toxin Internalization and Activation through Caveolae-like Domains

The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside G_M1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified, only 33% of surface-bound toxin was internalized by filipin-treated cells within 1 h compared with 79% in untreated cells. However, CT activation as determined by its reduction to form the A₁ peptide and CT activity as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding agent, 2-hydroxy-β-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization, activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not inhibit the latter, the two drugs appeared to distinguish between caveolae- and coated pit–mediated processes. In addition to its effects in CaCo-2 cells that express low levels of caveolin, filipin also inhibited CT activity in human epidermoid carcinoma A431 and Jurkat T lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely with alterations in the biochemical characteristics of CT-bound membranes due to the interactions of filipin with cholesterol rather than with the expressed levels of caveolin and caveolar structure. Our results indicated that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane rather than through a specific morphological structure and that these glycolipid microdomains have the necessary components required to mediate endocytosis.     

Harpin,An Elicitor of the Hypersensitive Response in Tobacco Caused by Erwinia amylovora,Elicits Active Oxygen Production in Suspension Cells

Active oxygen (AO) production and a K+/H+ exchange response (XR) are two concurrent early events associated with incompatible plant-bacteria interactions that result in a hypersensitive response (HR). Recently, a protein, termed harpin, produced by Erwinia amylovora has been reported to be the elicitor responsible for the HR caused by this pathogen. Although both the bacterium and harpin are reported to induce XR in tobacco (Nicotiana tabacum) cell suspensions, there have been no reports regarding the concurrent production of AO in this system. Here we report that E. amylovora stimulates the AO response, whereas an E. amylovora mutant that does not produce harpin does not elicit the AO response. In addition, a cell-free preparation of harpin induces AO production. This study indicates that harpin may be the bacterial elicitor of the XR and AO responses during the development of E. amylovora-induced HR.     

Steroid biochemistry and categorization of breast cyst fluid: relation to breast cancer risk

Patients bearing macrocysts of the breast are at higher risk of later developing cancer. The fluid filling the cysts (breast cysts fluid, BCF) contains unusual amounts of steroid conjugates, first androgen and estrogen sulfates. Measuring BCF cations (K⁺, Na⁺) allows categorization of cysts into two major subsets (type I and type II) that are associated with a different degree and/or turnover of apocrine metaplastic cells in the lining epithelium. Type I cysts (high K⁺/ Na⁺ ratio) accumulate huge amounts of dehydroepiandrosterone sulfate, estrone sulfate, androstane-3, 17β-diol glucuronide, androsterone glucuronide and contain more testosterone and dihydrotestosterone than type II. Conversely, type II cysts (low K⁺/Na⁺ ratio) contain more progesterone and pregnenolone. A cohort study was started in 1983 at the Cancer Prevention Center, Ravenna, Italy, with the aim of evaluating the relationships between the biochemistry of BCF and the incidence of breast cancer in women with gross cystic disease (GCD) of the breast. The bimodal distribution of the cationic pattern has been confirmed from data obtained in 798 patients aspirated. The risk of cyst relapse was significantly higher among women with type I cysts or with multiple cysts at presentation. Twelve incident cases of breast cancer have been diagnosed among women whose BCF was categorized. Eleven out of 12 cases had type I or multiple cysts. The cumulative incidence of breast cancer among patients bearing type I cysts was 2.5%. We conclude that women with GCD bearing type I cysts have an increased breast cancer risk when compared with the counterpart bearing type II cysts or the general population.