Date Palm DNA Mini-Preparation without Liquid Nitrogen

We have developed a new mini-procedure for isolation of total cellular DNA from date palm (Phoenix dactylifera L.). The procedure, which does not use liquid nitrogen, has proved useful due to temporary disruptions in supplies of liquid nitrogen that occur in countries where date palm trees are cultivated. DNA suitable for RFLP and PCR analyses is obtained.     

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.     

Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.     

The Macrophage Inhibitor CNI-1493 Blocks Metastasis in a Mouse Model of Ewing Sarcoma through Inhibition of Extravasation

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma.     

Dominance,Coloration, and Social and Sexual Behavior in Male Drills <Emphasis Type="Italic">Mandrillus leucophaeus</Emphasis>

Sexual selection has driven the evolution and elaboration of a wide variety of displays and ornaments in male nonhuman primates, including capes, cheek flanges, and sexual coloration. Among the most sexually dimorphic of all primates is the drill (Mandrillus leucophaeus), the males of which can be 3 times the mass of females, possess large canines, and exhibit extremely bright sexual skin coloration. However, the function of male coloration in this species has never been examined. Here, we present data on male color (measured objectively using digital photography), dominance rank, measures of male-female association, and key sexual behaviors, of adult male drills (n = 17) living in four semi free-ranging enclosures at the Drill Rehabilitation and Breeding Center in Nigeria. We test the hypothesis that male coloration is a badge of status, indicating dominance rank, and the hypothesis that male coloration attracts females. We found that male coloration did indicate rank, and that high ranking, strongly colored males were more likely to associate with adult females, and more specifically with fully tumescent females. These males also engaged in more sexual activity. However, measures of male-female association and sexual behaviors were not related to male color once rank had been taken into consideration; i.e., for males of a given rank, females did not prefer those that were more colorful. We discuss the results in light of what is known about the wild drill social system, in which unfamiliar individuals may come together in “supergroups,” and in which quality indicators of competitive ability may be particularly important.     

Spore age and coverslip placement affects germination and post-germination development of Colletotrichum dematium var circinans

Spore suspensions from young (10–14 da; young spores) and old (4 mo; old spores) colonies of PColletotrichum dematium var circinans were placed on slides. Coverslips were left off, placed on in the normal manner, or supported on shims. Slides were placed in moist chambers and incubated in light or dark for up to 48 hrs. Germination and post-germination development were studied. Shimming had some beneficial effect on germination, especially for old spores in dark. In general, more germ-tubes and appressoria were produced on spores under shims than spores with other coverslip treatments. By 48 hrs more old spores under shims germinated, and greater numbers of germ-tubes and appressoria were produced than on other old spores under different coverslip treatments. However, numbers produced were lower than those predicted for comparably treated young spores. Spore age, incubation regime, and placement of coverslips did not affect germ-tube initiation. For all treatments more germ-tubes were initiated from spore tops than bottoms or tips. Fewer germ-tubes were initiated from spore centers than other locations on tops and bottoms, and from both tips than one tip. Approximately 26 % of all appressoria were produced sessile. A higher percentage of sessile appressoria were produced on old spores (80 %) than on young spores (20%).     

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.