Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

Summary The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50% H₂O/CD₃OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The H ^N -H ^N sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The α-proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H ₂ ^α -H ^NEth is indicative of proximity between C- and N-termini.     

Microfacies and cycle stacking pattern in Liassic peritidal carbonate platform strata, Gavrovo-Tripolitza platform, Peloponnesus, Greece

Platform carbonate sediments of Liassic age cropping out in the area of the Pigadi-Fokianos Gulf (SE of Leonidion, Peloponnesus) have been investigated in order to determine their depositional environment. Facies analysis allowed the recognition of several microfacies types and their cyclic stacking pattern. The carbonates were deposited in a restricted inner platform environment (lagoon-peritidal domain) and are arranged into small-scale shallowing-upward cycles. Palaeosol horizons containing typical pedogenic features are developed on the top of the peritidal facies or are directly superimposed on subtidal deposits, forming diagenetic caps. This implies repeated sea-level fluctuations and periodic emersion episodes. The presence of orbitally forced cyclicity though is mostly probable, cannot be clearly documented by the available data. The studied carbonates are comparable with other coeval analogous peritidal cycles of the same age along the southern margin of the Tethys.     

Rapid evolution of yeast centromeres in the absence of drive

To find the most rapidly evolving regions in the yeast genome we compared most of chromosome III from three closely related lineages of the wild yeast Saccharomyces paradoxus. Unexpectedly, the centromere appears to be the fastest-evolving part of the chromosome, evolving even faster than DNA sequences unlikely to be under selective constraint (i.e., synonymous sites after correcting for codon usage bias and remnant transposable elements). Centromeres on other chromosomes also show an elevated rate of nucleotide substitution. Rapid centromere evolution has also been reported for some plants and animals and has been attributed to selection for inclusion in the egg or the ovule at female meiosis. But Saccharomyces yeasts have symmetrical meioses with all four products surviving, thus providing no opportunity for meiotic drive. In addition, yeast centromeres show the high levels of polymorphism expected under a neutral model of molecular evolution. We suggest that yeast centromeres suffer an elevated rate of mutation relative to other chromosomal regions and they change through a process of "centromere drift," not drive.     

Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin alphaIIbbeta3

Platelet aggregation requires agonist-induced alphaIIbbeta3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered alphaIIbbeta3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates alphaIIbbeta3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1-guanosine triphosphate-interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to alphaIIbbeta3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or beta3 that disrupt their mutual interaction block both talin recruitment and alphaIIbbeta3 activation. However, one talin mutant (L325R) is recruited to alphaIIbbeta3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated alphaIIbbeta3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and alphaIIbbeta3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to alphaIIbbeta3 by RIAM mediates agonist-induced alphaIIbbeta3 activation, with implications for hemostasis and thrombosis.     

Kinetics of Methyl t-Butyl Ether Cometabolism at Low Concentrations by Pure Cultures of Butane-Degrading Bacteria

Butane-oxidizing Arthrobacter (ATCC 27778) bacteria were shown to degrade low concentrations of methyl t-butyl ether (MTBE; range, 100 to 800 μg/liter) with an apparent half-saturation concentration (K_s) of 2.14 mg/liter and a maximum substrate utilization rate (k_c) of 0.43 mg/mg of total suspended solids per day. Arthrobacter bacteria demonstrated MTBE degradation activity when grown on butane but not when grown on glucose, butanol, or tryptose phosphate broth. The presence of butane, tert-butyl alcohol, or acetylene had a negative impact on the MTBE degradation rate. Neither Methylosinus trichosporium OB3b nor Streptomyces griseus was able to cometabolize MTBE.     

Stability of protein-encapsulating DRV liposomes after freeze-drying: A study with BSA and t-PA

Stability of protein-encapsulating DRV (dried-rehydrated vesicle) liposomes is evaluated after freeze-drying vesicles in presence (or not) of trehalose. Two proteins, bovine serum albumin (BSA) and tissue-type plasminogen activator (t-PA), are used, and protein-encapsulating liposomes with different lipid compositions are prepared by DRV technique. Encapsulation efficiencies are calculated, after measuring BSA with a fluorescence technique and t-PA's amidolytic activity toward a chromogenic substrate.Experimental results show that encapsulation of BSA in vesicles ranges between 35 and 53% of the protein and is only slightly affected by lipid composition. For t-PA, entrapment efficiencies are lower, ranging between 2 and 16%, while lipid composition has substantial effect on entrapment (cholesterol inclusion is very important). After freeze-drying, some lipid compositions remain stable, retaining most of initially entrapped proteins, while others do not, but they may be stabilized by trehalose. In the case of BSA, liposome behavior cannot be explained based on lipid membrane rigidity (more rigid = more stable). This may be connected with previously demonstrated interactions of BSA with membranes. Oppositely, t-PA behavior is more predictable, meaning that the lipid composition selected for the specific therapeutic application determines the need for cryoprotectant addition before freeze-drying t-PA containing DRV liposomes, perhaps due to the fact that under conditions applying minimum or no interactions between t-PA and lipid membranes occur.Thereby, interactions between proteins and membranes determine not only the encapsulation efficiency but also the need for cryopreservation of liposomal protein formulations.     

The effect of denture quality attributes on satisfaction and eating difficulties

Objectives: The development of a measure, with complimentary normative and perceived needs, for the investigation of functional impairment of individual patients and the investigation of the physical causative bases of impairment. Subjects: A consecutive sample of 119 complete denture (CD) wearers attending a Greek department of prosthetic dentistry took part in the study. All subjects presented with some complaint(s) of their dentures. Method: Functional assessment of dentures (FAD) criteria and structured questionnaires were used covering denture satisfaction (DS) and food difficulties with CDs. The questions used a five‐point Likert scale. Results: The results showed that there were statistically significant associations between satisfaction from dentures and eating specified foods. Statistically significant associations were also found between denture qualities and movement of upper dentures and difficulty eating specified foods, but not with an open question on the difficulty of eating unspecified foods. Conclusions: This study successfully validated a suitable model for the combined evaluation of denture quality with clear diagnostic criteria and closed questions on DS and perceived difficulties when eating specified foods. This could predict the need for treatment, monitor the impact of care and provide a feasible measure to meet the need of everyday clinical situations.     

Synthesis, characterization, and DNA adsorption studies of ampholytic model conetworks based on cross-linked star copolymers

Five model conetworks based on cross-linked star ampholytic copolymers were synthesized by group transfer polymerization. The ampholytic copolymers were based on two hydrophilic monomers: the positively ionizable 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the negatively ionizable methacrylic acid (MAA). Ethylene glycol dimethacrylate was used as the cross-linker. These five ampholytic model conetworks were isomers based on equimolar DMAEMA-MAA copolymer stars of different architectures: heteroarm (two), star block (two), and statistical. The two networks based on the homopolymer stars were also synthesized. The MAA units were introduced via the polymerization of tetrahydropyranyl methacrylate and the acid hydrolysis of the latter after network formation. All the precursors to the (co)networks were characterized in terms of their molecular weights using gel permeation chromatography (GPC). The mass of the extractables from the (co)networks was measured and characterized in terms of molecular weight and composition using GPC and proton nuclear magnetic resonance (1H NMR) spectroscopy, respectively. The degrees of swelling (DS) of all the ampholytic conetworks were measured as a function of pH and were found to present a minimum at a pH value which was taken as the isoelectric point, pI. The DS and the pI values did not present a dependence on conetwork architecture. Finally, DNA adsorption studies onto the ampholyte conetworks indicated that DNA binding was governed by electrostatics.