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Rheometric study of the gelation of chitosan in a hydroalcoholic medium.

The formation of chitosan physical hydrogels without any external cross-linking agent was studied. The gelation took place in an acetic acid-water-propanediol solution. The time to reach the gel point was determined by rheometry and gelations from different initial conditions could be compared. The influence of different parameters on gelation such as the polymer concentration, the degree of acetylation (DA) of chitosan and the composition of the initial solvent were investigated. The fractal morphology of the sample was not affected by the composition of the system. The number of junctions per unit volume at the gel point varied only with the initial number of chain entanglements per unit volume. Then, below an initial concentration of 1.5% (w/w), physical chain entanglements were insufficient and more junctions had to be formed to induce gelation. Over this value, only the kinetics allowing to replace entanglements by stable physical junctions played a key parameter. This kinetics was influenced by several parameters such as DA, temperature or the initial proportion water/alcohol. The acetyl groups played an important role in the formation of hydrophobic interactions, mainly responsible for gelation. The study of the influence of the gelation media revealed two critical points at 40% and 70% of water in the initial solvent, probably due to conformational changes and then to different modes of gelation. These physical hydrogels being used for cartilage regeneration, their final rheological properties were studied as a function of their degree of acetylation, the polymer concentration and the solvent composition in the initial solvent. Our results allowed us to define an optimal gelation condition for our application, corresponding to: DA=40%, a proportion water/alcohol of 50/50 and a polymer concentration of 1.5%.

Alcohols↗

Synthesis of double-headed 2-5A-antisense chimeras and their ability to activate human RNase L.

The synthesis of a novel 2-5A-antisense chimera having two molecules of a 2-5A tetramer at the 5'-terminus of the antisense moiety with a 2-(hydroxymethyl)-1,3-propanediol linker is described. The ability of the synthesized 2-5A antisense chimeras to activate RNase L was estimated by monitoring the cleavage of a target RNA by the activated RNase L. The double-headed 2-5A-antisense chimera linked with two molecules of a butanediol linker more efficiently cleaved the target RNA as compared with the single-headed 2-5A-antisense chimera and the double-headed 2-5A-antisense chimera linked with a molecule of the butanediol linker.

Adenine Nucleotides↗

Synthesis and biological properties of novel sphingosine derivatives.

Sphingosine-1-phosphate (S-1P) derivatives such as threo-(2S,3S)-analogues, which are C-3 stereoisomers of natural erythro-(2S,3R)-S-1P, have been synthesized starting from l-serine or (1S,2S)-2-amino-1-aryl-1,3-propanediols (6). threo-(1S,2R)-2-Amino-1-aryl-3-bromopropanols (HBr salt) have also been prepared from 6. The threo-S-1Ps and the threo-amino-bromide derivatives have shown potent inhibitory activity against Ca(2+) ion mobilization in HL60 cells induced by erythro-S-1P, suggesting that these compounds would compete with cell surface EDG/S1P receptors.

Calcium Signaling↗

Structure of dihydroxyacetone phosphate dimethyl acetal, a stable dihydroxyacetone phosphate precursor, in the crystalline state.

Crystal and molecular structures of four different salts of a dihydroxyacetone phosphate (DHAP) precursor, its dimethyl acetal [2,2-dimethoxy-1,3-propanediol phosphate, C(5)H(13)O(7)P, (MeO)(2)DHAP]: (cha)(2)[(MeO)(2)DHAP].H(2)O (6a), (cha)[(MeO)(2)DHAP] (6b), Na(2)[(MeO)(2)DHAP].5.75H(2)O (6c) and K(2)[(MeO)(2)DHAP].H(2)O (6d), along with the cyclohexylammonium (cha) salt of its phenyl ester (cha)[(MeO)(2)DHAP(Ph)] (6e) are described. In the (MeO)(2)DHAP mono- and dianions, slightly different orientation of the phosphate group in relation to the acetal carbon atom is observed, with a delicate tendency of phosphate group to be located antiperiplanar in the monoanions and anticlinal in the dianions. The 2,2-dimethoxy-1,3-propandiol moiety, (MeO)(2)DHA, seems to be very rigid and its conformation is independent of phosphorylation, the ionization state of the inserted phosphate group and its additional substitution. The overall structures of the cyclohexylammonium (6a,b) and potassium salts (6d) have a double-layered architecture, while the sodium cation network in 6c forms the system of channels, which are filled up with the [(MeO)(2)DHAP](2-) ions. The different architectures of 6c and 6d crystals result from the different ways in which the relevant dianions coordinate to sodium and potassium ions and affect also the hydrogen bonding system observed in 6c and 6d crystals.

Acetals↗

Cryobanking of fish somatic cells: optimizations of fin explant culture and fin cell cryopreservation.

When gametes or embryos are not available, somatic cells should be considered for fish genome cryobanking of valuable or endangered fish. The objective of this work was to develop a method for fin explant culture with an assessed reliability, and to assess fin cells ability to cryopreservation. Anal fins from goldfish (Carassius auratus) were minced and gently loosened with collagenase before explants were plated at 20 degrees C in L-15 medium supplemented with fetal bovine serum and pH buffering additives. Quantification of cell-donor explants per fin rated the culture success. Cells were successfully obtained from every cultured anal fin (mean = 65% cell-donor explant per fin). All other fin types were suitable except the dorsal fin. Explant plating could be deferred 3 days from fin collecting. Fins from seven other fish species were successfully cultured with the method. After 2-3 weeks, sub-confluent fin cells from goldfish were cryopreserved. Cryopreservation with dimethyl sulfoxide and sucrose at a slow freezing rate allowed the recovery of half the goldfish fin cells. Cells displayed the same viability as fresh ones. 1,2-propanediol was unsuitable when a fast freezing rate was used. The procedure could now be considered for cryobanking with only minimal adaptation to each new species.

Animals↗

Insights into the retention mechanism of neutral organic compounds on polar chemically bonded stationary phases in reversed-phase liquid chromatography.

The solvation parameter model is used to characterize the retention properties of a 3-aminopropylsiloxane-bonded (Alltima amino), three 3-cyanopropylsiloxane-bonded (Ultrasphere CN, Ultremex-CN and Zorbax SB-CN), a spacer bonded propanediol (LiChrospher DIOL) and a multifunctional macrocyclic glycopeptide (Chirobiotic T) silica-based stationary phases with mobile phases containing 10 and 20% (v/v) methanol-water. The low retention on the polar chemically bonded stationary phases compared with alkylsiloxane-bonded silica stationary phases arises from the higher cohesion of the polar chemically bonded phases and an unfavorable phase ratio. The solvated polar chemically bonded stationary phases are considerably more hydrogen-bond acidic and dipolar/polarizable than solvated alkylsiloxane-bonded silica stationary phases. Selectivity differences are not as great among the polar chemically bonded stationary phases as they are between the polar chemically bonded phases and alkylsiloxane-bonded silica stationary phases.

Chromatography, Liquid↗

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model. III. Polar stationary phases.

In this third paper, varied types of polar stationary phases, namely silica gel (SI), cyano (CN)- and amino-propyl (NH2)-bonded silica, propanediol-bonded silica (DIOL), poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), were investigated in subcritical fluid mobile phase. This study was performed to provide a greater knowledge of the properties of these phases in SFC, and to allow a more rapid and efficient choice of polar stationary phase in regard of the chemical nature of the solutes to be separated. The effect of the nature of the stationary phase on interactions between solute and stationary phases and between solute and carbon dioxide-modifier mobile phases was studied by the use of a linear solvation energy relationship (LSER), the solvation parameter model. The retention behaviour observed with sub/supercritical fluid with carbon dioxide-methanol is close to the one reported in normal-phase liquid chromatography with hexane. The hydrogen bond acidity and basicity, and the polarity/polarizability favour the solute retention when the molar volume of the solute reduces it. As with non-polar phases, the absence of water in the subcritical fluid allows the solute/stationary phase interactions to play a greater part in the retention behaviour. As expected, the DIOL phase and the bare silica display a similar behaviour towards acidic and basic solutes, when interactions with basic compounds are lower with the NH2 phase. On the CN phase, all interactions (hydrogen bonding, dipole-dipole and charge transfer) have a nearly equivalent weight on the retention. The polymeric phases, PEG and PVA, provide the most accurate models, possibly due to their better surface homogeneity.

Carbon Dioxide↗

Chemical and chromatographic stability of methacrylate-based monolithic columns.

Chemical and chromatographic stability of methacrylate-based monolithic columns bearing 3-N,N-diethylamino-2-hydroxypropyl (DEAE) and quarternary amine (QA) groups was studied. The leakage products from both monolithic columns were determined and the leakage of amines has been quantified in alkali solutions. Monolithic columns bearing QA functional groups being exposed to 1M sodium hydroxide solution for up to 3 months caused reduction of ion-exchange groups for approximately 12%, while for DEAE monolithic columns was only around 3% in 1 year. In 0.1M NaOH and 20% ethanol degradation was significantly lower. The main leaking compound from DEAE monolith was found to be 3-(diethylamino)-1,2-propanediol and 2,3-dihydroxypropyltrimethylammonium salt for QA monolith. During repeated 50 cleaning-in-place (CIP) cycles, no changes in chromatographic properties were detected.

Chromatography, Ion Exchange↗

Solution-state NMR investigation of DNA binding interactions in Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg): a dynamic description of the DNA/protein interface.

Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair (BER) protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free in solution and bound to a double-stranded DNA oligomer containing 1,3-propanediol (13-PD), a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 251 (83%) free-precession 15N/1H HSQC cross peaks were observed and 180 of these were assignable, indicating that approximately 30% of the residues undergo intermediate motion on the NMR timescale, broadening the resonances beyond detection or making them intractable in backbone assignment experiments. The majority of these affected residues were in the polypeptide linker region and the interface between the N- and C-terminal domains. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate timescale motion observed for free Fpg, including those residues directly involved in DNA binding, notwithstanding a nanomolar dissociation constant for 13-PD binding. Indeed, after binding to 13-PD, at least approximately 40% of the Fpg residues undergo intermediate timescale motion even though all other residues exhibit tight DNA binding characteristic of slow exchange. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that were quenched upon binding 13-PD. In free Fpg, heteronuclear 1H-15N NOE experiments detected picosecond timescale backbone motion in the alphaF-beta9 loop, the region primarily responsible for chemically discriminating 8-oxoguanine (8-oxoG) over normal guanine, that was quenched after binding 13-PD. Collectively, these observations reveal that, in solution, Fpg is a very dynamic molecule even after binding damaged DNA. Such motion, especially at the DNA binding surface, may be key to its processive search for DNA damage and its catalytic functions once it recognizes damaged DNA.

Amino Acid Sequence↗

Cryopreservation of human ovarian tissue.

Approaches to the cryopreservation of human ovarian tissue are typically characterised by the use of slow freezing/rapid thawing methods using dimethyl sulfoxide or 1,2-propanediol (PROH) as cryoprotectants. This paper reviews current experience with these procedures.

Cryopreservation↗

Felbamate is a subunit selective modulator of recombinant gamma-aminobutyric acid type A receptors expressed in Xenopus oocytes.

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is clinically available for the treatment of refractory epileptic seizures, and is known to modulate several ion channels including gamma-aminobutyric acid type A (GABA(A)) receptors. To determine felbamate subunit selectivity for GABA(A) receptors we expressed 15 different GABA(A) receptor combinations in Xenopus laevis oocytes. Felbamate positively modulated GABA-currents of alpha(1)beta(2)gamma(2S), alpha(1)beta(3)gamma(2S), alpha(2)beta(2)gamma(2S) and alpha(2)beta(3)gamma(2S), whereas felbamate was either ineffective or negatively modulated the other 11 receptor combinations. Regional distributions of GABA(A) receptor subunits suggest that felbamate may differentially modulate distinct inhibitory circuits, a possibility that may have relevance to felbamate efficacy in refractory epilepsies.

Animals↗

Pregnancies and births after oocyte cryopreservation.

OBJECTIVE: To determine the potential of oocyte cryopreservation techniques. DESIGN: Retrospective data analysis. SETTING: A tertiary infertility center. PATIENT(S): Sixty-eight patients (29 to 37 years of age) undergoing assisted reproduction procedures for infertility problems. INTERVENTION(S): Oocytes from women treated for infertility were cryopreserved with a slow cooling/rapid thawing protocol in which 1,2 propanediol and sucrose were used as cryoprotectants. Eighty-six thawing cycles were performed. MAIN OUTCOME MEASURE(S): Rates of survival after thawing, fertilization after intracytoplasmic sperm injection, cleavage, implantation, and pregnancy. RESULT(S): We treated 68 patients through 86 thawing cycles. Seven hundred thirty-seven oocytes were thawed, and 59 transfer cycles were performed. The survival rate was 37%. The fertilization and cleavage rates were 45.4% and 86.3%, respectively. A total of 15 clinical pregnancies were achieved with pregnancy rates of 25.4% per transfer and 22% per patient. There were three miscarriages, resulting in an abortion rate of 20%. Seventeen of the 104 transferred embryos implanted, corresponding to an implantation rate of 16.4%. Thirteen babies were born, 8 females and 5 males. CONCLUSION(S): Statistically significant results were obtained for fertilization, cleavage, and pregnancy rates. Our results show oocyte cryopreservation may represent an alternative to embryo storage in selected cases.

Cell Survival↗

Birth of a healthy baby after transfer of blastocysts derived from cryopreserved human oocytes fertilized with frozen spermatozoa.

OBJECTIVE: To report the birth of a healthy baby after transfer of blastocysts derived from frozen eggs and frozen spermatozoa. DESIGN: Case report. SETTING: University-based assisted reproduction center. PATIENT(S): A 37-year-old woman with secondary infertility of 4 years' duration. INTERVENTION(S): Retrieved oocytes were cryopreserved in 1.5 M 1,2-propanediol (PROH) and 0.3 M sucrose by a slow freezing-rapid thawing protocol, semen cryopreservation, and insemination by intracytoplasmic sperm injection (ICSI) at 4 hours after thawing. MAIN OUTCOME MEASURE(S): Fertilization and embryo development to blastocyst stage, pregnancy, and outcome. RESULT(S): Ten of 14 frozen oocytes survived after thawing. Eight of them were fertilized by performing ICSI and three developed into 7- to 8-cell embryos on day 3. Two of these embryos developed into blastocysts on day 5 and were transferred. This resulted in a successful pregnancy and the delivery of a healthy baby boy. CONCLUSION(S): This case demonstrates the feasibility of inseminating the frozen-thawed human oocytes after 4 hours of in vitro culture and the zygotes derived from frozen oocytes and frozen spermatozoa can be cultured to blastocysts resulting in the live birth of a healthy baby boy.

Adult↗

Human oocyte cryopreservation and the fate of cortical granules.

OBJECTIVE: To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. DESIGN: Laboratory study. SETTING: IVF unit. INTERVENTION(S): Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing-rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. MAIN OUTCOME MEASURE(S): Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). RESULT(S): The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. CONCLUSION(S): The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes.

Cells, Cultured↗

Report of four donor-recipient oocyte cryopreservation cycles resulting in high pregnancy and implantation rates.

OBJECTIVE: To determine the clinical potential of donor-oocyte cryopreservation and thaw techniques for recipient patients. DESIGN: Institutional review board-approved prospective study of donor oocyte cryopreservation. SETTING: A large, private infertility center. PATIENT(S): Four anonymous oocyte donors underwent ovarian hyperstimulation for the purpose of oocyte retrieval and cryopreservation. The oocytes were subsequently thawed, fertilized, and transferred to 4 recipient patients. INTERVENTION(S): Oocytes were obtained from young donor patients and were cryopreserved with a slow freeze/rapid thaw protocol in which 1,2-propanediol (PrOH) and sucrose were used as cryoprotectants. Oocytes that survived were inseminated using intracytoplasmic sperm injection (ICSI). Resulting embryos were replaced into the recipient patients on the third day post-insemination. MAIN OUTCOME MEASURE(S): Post-thaw survival rate, fertilization rate, cleavage rate, implantation and clinical pregnancy rates. RESULT(S): A total of 79 metaphase II oocytes were frozen, stored frozen overnight in liquid nitrogen, and then thawed. The post-thaw survival rate was 86.1%. Normal fertilization following ICSI occurred in 89.7% of the surviving oocytes. Cleavage was observed in 91.8% of normally fertilized oocytes. A total of 23 embryos were transferred to 4 recipient patients. A clinical pregnancy rate of 75% and an implantation rate of 26.1% were achieved. CONCLUSION(S): Human oocyte cryopreservation is an effective technique that can be applied in clinical situations with high oocyte survival and clinical pregnancy rates expected.

Adult↗

Specific regions of genome plasticity and genetic diversity of the commensal Escherichia coli A0 34/86.

Escherichia coli A0 34/86 (O83:K24:H31) is a commensal strain that has been used for prophylactic and therapeutic colonization of the intestine of newborn infants. To identify traits specific for E. coli A0 34/86, we used a minimal tiling set of 148 BAC clones of A0 34/86 genomic DNA, to construct restriction-digested BAC arrays. Hybridization with genomic DNA from four E. coli strains (CFT073; O157:H7; K12 and Nissle 1917) allowed selection of two BAC clones that were sequenced to identify A0 34/86-specific regions. Genes for the yersiniabactin siderophore system, several proteins homologous to Salmonella enterica serovar Typhimurium vitamin B12 synthesis proteins, as well as genes necessary for the degradation of propanediol, the pix fimbriae determinant and genes coding for a putative phosphoglycerate transport system present also on pathogenicity island V of E. coli strain 536 were all identified in E. coli A0 34/86. This comparative analysis underlines the important genome heterogeneity between E. coli strains.

Chromosomes, Artificial, Bacterial↗

ATR-FTIR characterization of transport properties of benzoic acid ion-pairs in silicone membranes.

A novel technique based on Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to study the transport of benzoic acid ion-pairs/salts in silicone membranes. The benzoic acid ion-pairs were prepared using various counter-ions with different degrees of lipophilicity, e.g. triethylamine (TA), diethylamine (DE), tert-butylamine (t-BA), 2-amino-2-methyl-propanol (AMP), and 2-amino-2-methyl-propanediol (AMPD). Silicone membrane, treated or untreated with propylene glycol (PG), was placed on the surface of a ZnSe crystal and the transport solution was applied to the upper surface of the membrane. A mathematical model, based on Fick's second law describing the build up of permeant concentration at the membrane/crystal interface with time was applied to determine diffusion coefficients. Absorption due to the acid (1700 cm(-1)) or benzoate anion (1555 cm(-1)) was observed at different regions without the interference from PG or silicone membrane. Benzoate anion, a charged species, was observed to permeate the membrane. The permeation of benzoate anion from sodium benzoate and polar ion-pairs of AMP and AMPD was very low in contrast to their high-saturated concentrations in PG as compared to the t-BA ion-pair. This indicated that benzoate anion preferentially permeates the membrane as an ion-pair rather than a single anion; otherwise its permeation should correspond to its concentration in PG instead of the lipophilicity of the ion-pairs. Additionally, the diffusion coefficient values of benzoic acid and benzoate anions through the treated and untreated membranes were not statistically different.

Antifungal Agents↗

Discovery and investigation of a new, second triose phosphate isomerase in Klebsiella pneumoniae.

In this study, a tpi1 gene encoding for the enzyme triose phosphate isomerase in Klebsiella pneumoniae DSM2026 was knocked out in an effort to metabolically engineer this strain as a model system for the production of 1,3-propanediol. Investigations of the tpi1 knockout mutant led to the discovery of a second tpi gene (tpi2) in this organism. The new tpi2 gene was cloned and sequenced. The coding region of the tpi2 gene contains 795bp (base pairs) and the deduced protein consists of 265 amino acids. Sequence comparison of TPI2 proteins in different organisms revealed the presence of a highly conserved signature A-Y-E-P-V-W-A-I-G-[EDVS]-[GKNASH], which is nearly the same as the reported TPI consensus signature. The tpi1 gene of K. pneumoniae DSM2026 shows a high sequence similarity to that of E. coli, whereas, the tpi2 gene resembles more its relatives in the alpha-proteobacteria, suggesting that they evolve from different ancestors. The overexpression of the tpi2 gene restores the growth deficiency of tpi1 knockout mutant on the minimal medium containing glucose or glycerol. Furthermore, the catalytic activity of this new triose phosphate isomerase was confirmed in both tpi1 knockout mutant and tpi2 over-expressing strain by enzyme assays. For the first time, the co-existence of two tpi genes in an enteric bacterium is experimentally confirmed.

3' Flanking Region↗