Reproductive lability in pre-dispersal biology ofRhamnus alaternus L. (Rhamnaceae)

Summary Rhamnus alaternus L. is a dioecious, fleshy-fruited shrub, typical of Mediterranean vegetation, which has been recorded from the region since the tropical Tertiary. Reproductive biology of this species has been studied in Southern Italy over a period of 4 years with both field and laboratory experiments. In all the studied populations male plants predominate over the females, have a higher flower density and flower regularly every year while females are usually alternate-bearing. A strong negative association between fruit formation and vegetative growth has been found indicating that the presence of fruits on a branch reduces the growth of new shoots. This phenomenon, together with alternate-bearing, is evidence of a strong competition between vegetative growth and reproductive activity and indicates resource limitation. Flower morphology and quantity of pollen produced are typical of insect pollinated species, and flies of the Empididae and Muscidae were trapped bearing pollen at female plants but pollen is also dispersed by wind. Field experiments on pollination success showed that the exclusion of insects as pollen vectors produces a significant decrease in fruit development. Excess pollen provided by hand-pollination increases fruit set over open-pollination and reproductive success in the field is therefore pollen limited. The variability of sex expression, male fitness, fruit production, regrowth and pollination presented here suggests that these predispersal characteristics ofR. alaternus have been subject to evolutionary change since the tropical Tertiary climate was replaced by a Mediterranean one. This points to the possibility that reproductive, as well as morphological and physiological characteristics, may be equally selected for in extreme environments such as the Mediterranean.     

Synthesis and comparative conformational energetics of D-phenylalanylsarcosine and its cyclic dehydration product, (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione

Summary Synthetic protocols are presented both for D-PheSar and the corresponding cyclised diketopiperazine, prepared from N-t-butoxycarbonylprotected D-PheSar. Deprotection conditions could be manipulated to yield either D-Phenylalanylsarcosine or (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione. Molecular modelling revealed several low energy conformers which contained a Z-peptide bond and which were readily amenable to cyclisation. Cyclisation was found by HPLC to be fastest in strongly acidic conditions.Abbreviation HBTU o-Benzotriazolyl-tetramethyluronium hexafluorophosphate     

Nutrient ratios, differential retention, and the effect on nutrient limitation in a deep oligotrophic lake

Nutrient ratios have been related to nutrient limitation of algal growth in lakes. Retention of nutrients in lakes, by sedimentation and by denitrification, reduces the nutrient concentrations in the water column, thereby enhancing nutrient limitation. Differential retention of nitrogen and phosphorus alters their ratios in lakes and thereby contributes to determine whether nitrogen or phosphorus limits algal growth. We examined the relationships between differential nutrient retention, nutrient ratios, and nutrient limitation in Lake Brunner, a deep oligotrophic lake. The observed retention of nitrogen (20%) and phosphorus (47%) agreed with predictions by empirical equations from literature. As a result of differential retention with a much larger proportion of phosphorus retained than that of nitrogen, the nitrogen:phosphorus ratio was higher in the lake (69) than in the inflows (46). While the mean ratio in the inflows suggested no or only moderate phosphorus limitation, the lake appeared to be severely phosphorus limited. Combining empirical equations from literature that predict nitrogen and phosphorus retention suggests that the nitrogen:phosphorus ratio is enhanced by greater retention of phosphorus compared to nitrogen only in deep lakes with relatively short residence times, such as Lake Brunner. In contrast, in most lakes differential retention is expected to result in lower nitrogen:phosphorus ratios.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Brazilian Angiostrongylus cantonensis haplotypes,ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.     

Neuromuscular Electrical Stimulation of the Quadriceps in Patients with Non-Small Cell Lung Cancer Receiving Palliative Chemotherapy: A Randomized Phase II Study

Background

A reduced exercise capacity is associated with increased morbidity and mortality in patients with advanced non-small cell lung cancer (NSCLC). Therapeutic exercise can be beneficial and neuromuscular electrical stimulation (NMES) of the quadriceps muscles may represent a practical approach. The primary aim of this study was to determine the acceptability of NMES of the quadriceps to patients with NSCLC used alongside palliative chemotherapy. Secondary aims explored aspects of safety and efficacy of NMES in this setting.

Methods

Patients with advanced NSCLC due to receive first-line palliative chemotherapy were randomized to usual care with or without NMES. They were asked to undertake 30 minute sessions of NMES, ideally daily, but as a minimum, three times weekly. For NMES to be considered acceptable, it was predetermined that ≥80% of patients should achieve this minimum level of adherence. Qualitative interviews were held with a subset of patients to explore factors influencing adherence. Safety was assessed according to the Common Terminology Criteria for Adverse Events. Quadriceps muscle strength, thigh lean mass, and physical activity level were assessed at baseline and after three cycles of chemotherapy.

Results

49 patients (28 male, median (IQR) age 69 (64−75) years) participated. Of 30 randomized to NMES, 18 were eligible for the primary endpoint, of whom 9 (50% [90% CI, 29 to 71]) met the minimum level of adherence. Adherence was enhanced by incorporating sessions into a daily routine and hindered by undesirable effects of chemotherapy. There were no serious adverse events related to NMES, nor significant differences in quadriceps muscle strength, thigh lean mass or physical activity level between groups.

Conclusions

NMES is not acceptable in this setting, nor was there a suggestion of benefit. The need remains to explore NMES in patients with cancer in other settings.

Trial Registration

Current Controlled Trials ISRCTN 42944026 www.controlled-trials.com/ISRCTN42944026     

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2- 8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.      

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.