Evidence for cooperative effects in the binding of polyvalent metal ions to pure phosphatidylcholine bilayer vesicle surfaces

An internal NMR monitor for the study of lanthanide ion (Ln³⁺) binding to phospholipid bilayer membranes has been developed. The dimethylphosphate anion, DMP^?, forms labile complexes with Ln³⁺ in aqueous solution and in solutions also containing bilayer dispersions. The hyperfine shift in the DMP^? resonance induced by Pr³⁺ ions has been used to determine the overall thermodynamic formation constants for the Pr(DMP)²⁺ and Pr(DMP)₂⁺ complexes: 81 (M^?1) and 349 (M^?2) at 52°C; the limiting hyperfine shift (³¹P) at 52°C is 91.5 ppm downfield. These parameters, applied to the observed DMP^? hyperfine shift in the presence of the membrane, establish both the free Pr³⁺ concentration and the amount of Pr³⁺ bound to the phospholipid surface. Extensive data for the binding of Pr³⁺ to the outer surfaces of sonicated vesicles yield a limiting hyperfine shift per Pr³⁺ of 181.6 ppm downfield for the dipalmitoylphosphatidylcholine ³¹P resonance at 52°C, clearly demonstrating that the binding stoichiometry is two DPPCs per Pr³⁺. A Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherm incorporating electrostatic considerations at the Debye-Hückel level. Fittings to specific models lead to a cooperative model in which tense (T) sites, with low affinity for Pr³⁺, present in the absence of metal ions, quickly give way to relaxed (R) sites (two DPPCs per site), with much higher affinity for Pr³⁺, as the amount of Pr³⁺ bound to the surface increases. The intrinsic equilibrium constants for the binding of Pr³⁺ to DPPC vesicles are 2 M^?1 and 3 000 M^?1 for the T and R sites, respectively, at 52°C. The distribution coefficient between these sites ([R]/[T]) in the absence of Ln³⁺ is 0.14 at 52°C. We picture the binding site conversion as a head-group conformational change involving mostly the choline moiety. Sketchy results for binding on the inside vesicle surface indicate that the overall affinity for Pr³⁺ is significantly greater and suggest that the site stoichiometry may be different.     

Src kinases mediate VEGFR2 transactivation by the osteostatin domain of PTHrP to modulate osteoblastic function

Parathyroid hormone‐related protein (PTHrP) stimulates osteoblastic function through its N‐ and C‐terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107–139) and the shorter PTHrP (107–111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3‐E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr‐1059 within 5–10 min in both MC3T3‐E1 and rat osteoblastic osteosarcoma UMR‐106 cells. This phosphorylation elicited by osteostatin appears to be VEGF‐independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal‐regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin‐dependent induction of ERK and Akt activation was abrogated by SU6656. Up‐regulation of VEGF and osteoprotegerin gene expression as well as the pro‐survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C‐terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function. J. Cell. Biochem. 114: 1404–1413, 2013. © 2012 Wiley Periodicals, Inc.     

SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.     

Assemblage structure,vertical distributions and stable-isotope compositions of anguilliform leptocephali in the Gulf of Mexico

In August 2007, October 2008 and September–October 2010, 241 Tucker trawl and plankton net tows were conducted at the surface to depths of 1377 m at six locations in the northern and eastern Gulf of Mexico (GOM) to document leptocephalus diversity and determine how assemblage structure, larval size, abundance and isotopic signatures differ across the region and with depth. Overall, 2696 leptocephali representing 59 distinct taxa from 10 families were collected. Five families accounted for 96% of the total catch with Congridae and Ophichthidae being the most abundant. The top four most abundant species composed 59% of the total catch and included: Ariosoma balearicum, Paraconger caudilimbatus, Rhynchoconger flavus and Ophichthus gomesii. Four anguilliform species not previously documented in the GOM as adults or leptocephali were collected in this study, including Monopenchelys acuta, Quassiremus ascensionis, Saurenchelys stylura and one leptocephalus only known from its larval stage, Leptocephalus proboscideus. Leptocephalus catches were significantly greater at night than during the day. Catches at night were concentrated in the upper 200 m of the water column and significantly declined with increasing depth. Leptocephali abundances and assemblages were significantly different between sites on the upper continental slope (c. 500 m depth) and sites on the middle to lower continental slope (c. 1500–2300 m). Sites on the lower continental slope had a mixture of deep-sea demersal, bathypelagic and coastal species, whereas upper-slope sites contained several numerically dominant species (e.g., A. balearicum, P. caudilimbatus) that probably spawn over the continental shelf and upper slope of the GOM. Standard lengths of the four dominant species differed between sites and years, indicating heterochronic reproduction and potential larval source pools within and outside of the GOM. Stable-isotope analyses (δ¹³C and δ¹⁵N) conducted on 185 specimens from six families revealed that leptocephali had a wide range of isotopic values at the family and size-class levels. Species in the families Muraenidae, Congridae and Ophichthidae had similar δ¹⁵N values compared with the broad range of δ¹⁵N values seen in the deep-sea families Nemichthyidae, Nettastomatidae and Synaphobranchidae. Stable-isotope values were variably related to length, with δ¹⁵N values being positively size correlated in ophichthids and δ¹³C values being negatively size correlated in A. balearicum and P. caudilimbatus. Results suggest that leptocephali feed in various water depths and masses, and on different components of POM, which could lead to niche partitioning. Ecological aspects of these important members of the plankton community provide insight into larval connectivity in the GOM as well as the early life history of Anguilliformes.     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.     

Patterns of care of radiotherapy in México

Aim

This survey is performed to learn about the structure of radiotherapy in México.

Background

Radiation oncology practice is increasing because of the higher incidence of cancer. There is no published data about radiotherapy in México.

Materials and methods

A questionnaire was sent to the 83 registered centers in the database of the Mexican regulatory agency. One out of the 32 states has no radiotherapy. 27 centers from 14 states provided their answers.

Results

829 patients are treated annually with any radiotherapy modality in each center. Two centers have one cobalt machine, 7 have a cobalt and a linac and 10 have more than one linac. Five centers use 2D planning systems, 22 use 3D; 9, conventional simulators; 22, CT based simulation, and 1 center has no simulation. Most of the centers verify beams with films, electronic portal image devices and cone beam CTs are also used. Intensity modulated and image guided radiotherapy are performed in 5 states. Breast, prostate, cervix, lung, rectum and head and neck cancer are the six most common locations. There are 45 public and 38 private centers, 2 dedicated to children. Two gamma knife units, 5 Novalis systems, 1 tomotherapy and 2 cyberknife machines are working. All centers have at least one radiation oncologist, one physicist and one radiotherapist.

Conclusions

Definitive conclusions cannot be drawn from this limited feedback due to a low participation of centers. This survey about radiotherapy in Mexico shows the heterogeneity of equipment as well as medical and technical staff in the whole country.     

Vitamin D Status: A Different Story in the Very Young versus the Very Old Romanian Patients

BackgroundIn Romania (latitude 48°15’N to 43°40’N), vitamin D supplementation is common practice mostly in infants 0-1 year old. No published information is available regarding epidemiological data on vitamin D status in the Romanian population for a wide age range and geographical territory. In this context, we aimed to evaluate the seasonal and age variation of vitamin D status in a large Romanian population.Methods6631 individuals from across Romania had performed 7544 vitamin D assessments (2012-2014) in a chain of private laboratories. Vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) was measured using High Performance Liquid Chromatography. Vitamin D levels were classified as severe deficiency<10ng/mL, deficiency 10-20ng/mL, insufficiency 21-29ng/mL, sufficiency≥30ng/mL and potentially harmful>100ng/ml.ResultsMale to female ratio was 1:2.9. Age ranged from 0 to 85 years. Mean vitamin D levels increased from April (26.3ng/ml) to September (35.6ng/ml) and decreased from October (33.5ng/ml) to March (24.4 ng/ml). Overall 40% had sufficient vitamin D, while the rest were insufficient 33%, deficient 22%, severely deficient 4% and 1% potentially harmful (of them 81% under 1 year old). Males compared to females showed higher percentages of sufficiency (47% vs. 38%). Children 0- 2 years presented the highest percentage of vitamin D sufficiency (77%). Lowest percentages (21%) of sufficiency were in people 80-84 years.ConclusionIn Romania, suboptimal vitamin D levels are common (59%), especially in older age, wintertime and in women. Vitamin D supplementation would be most warranted from January to April in the Romanian population. 25-hydroxyvitamin D levels>100ng/ml were relatively prevalent in children 0-1 year old (17.3%). This was attributed to supplementation errors and the fact that high-risk individuals were more likely to visit for medical check-up. Nonetheless, it stresses the need to increase awareness of the importance of preventing Vitamin D supplementation administration errors in the young.     

Cytotoxic palladium complexes of bioreductive quinoxaline N1,N4-dioxide prodrugs

Four new palladium(II) complexes with the formula Pd(L)₂, where L are quinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives, were synthesized as a contribution to the chemistry and pharmacology of metal compounds with this class of pharmacologically interesting bioreductive prodrugs. Compounds were characterized by elemental, conductometric and thermogravimetric analyses, fast atom bombardment mass spectrometry (FAB-MS) and electronic, Fourier transform infrared (FTIR) and ¹H-nuclear magnetic resonance spectroscopies. The complexes were subjected to cytotoxic evaluation on V79 cells in hypoxic and aerobic conditions. In addition, a preliminary study on interaction with plasmid DNA in normoxia was performed. Complexes showed different in vitro biological behavior depending on the nature of the substituent on the quinoxaline ring. Pd(L1)₂ and Pd(L2)₂, where L1 is 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide and L2 is 3-amino-6(7)-methylquinoxaline-2-carbonitrile N¹,N⁴-dioxide, showed non selective cytotoxicity, being cytotoxic either in hypoxic or in aerobic conditions. On the other hand, Pd(L3)₂, where L3 is 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N¹,N⁴-dioxide, resulted in vitro more potent cytotoxin in hypoxia (P = 5.0 μM) than the corresponding free ligand (P = 9.0 μM) and tirapazamine (P = 30.0 μM), the first bioreductive cytotoxic drug introduced into clinical trials. In addition, it showed a very good selective cytotoxicity in hypoxic conditions, being non-cytotoxic in normoxia. Its hypoxic cytotoxicity relationship value, HCR, was of the same order than those of other hypoxia selective cytotoxins (i.e., Mitomycine C, Misonidazole and the N-oxide RB90740). Interaction of the complexes with plasmid DNA in normoxia showed dose dependent ability to relax the negative supercoiled forms via different mechanisms. Pd(L2)₂ introduced a scission event in supercoiled DNA yielding the circular relaxed form. Meanwhile, both Pd(L1)₂ and Pd(L3)₂ produced the loss of negative supercoils rendering a family of topoisomers with reduced electrophoretic mobility. Pd(L3)₂ showed a more marked effect than Pd(L1)₂. Indeed, for the highest doses assayed, Pd(L3)₂ was even able to introduce positive supercoils on the plasmid DNA.     

Aspects of interspecific hybridization within edible <Emphasis Type="Italic">Alliaceae</Emphasis>

Crossing experiments within the genus Allium have been carried out for many years. Usually, with the aim of widening genetic variation, edible Alliaceae such as bulb onion, Japanese bunching onion, leek, garlic and shallot have been crossed with each other or with distant vegetable Allium crops. Interspecific hybridization, especially with wild relatives is considered as the best way of introgression desirable traits to the crops. By using sexual or somatic hybridization, many important characters, such as: disease and pest resistance, important metabolites and cytoplasmic male sterility have been transferred to other Allium crops. This review summarises some aspects of the directions of interspecific hybridization in edible Alliaceae and the significance and perspectives of using these hybrids in breeding programmes.