Physical-Chemical Basis of the Protection of Slowly Frozen Human Erythrocytes by Glycerol

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. (Cryobiology, 13:9-23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur (Biophys. J., 23:89-100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT₅₀) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT₅₀ for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT₅₀ corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: (a) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.     

Synthesis of an α-kojibiosyl substituted glycerol teichoic acid hexamer

In this paper the synthesis of an Enterococcus Faecalis teichoic acid (TA) hexamer is presented. The key kojibiosyl-glycerol phosphoramidite building block was obtained by condensation of thioglucose donors, provided with various protecting groups at the C2 hydroxyl function with an orthogonally protected glycerol acceptor. After selective deprotection, the resulting 1,2-cis-linked pseudodisaccharide acceptor was coupled to an α-directing thioglucose donor, giving the corresponding pseudotrisaccharide, which is then transformed to a phosphoramidite synthon. The kojibiosyl-glycerol phosphoramidite in combination with a glycerolphosphoramidite, an aminohexylphosphoramidite and dibenzylglycerol were coupled to a fully protected glycerol TA hexamer, using chemistry that can be amended for future automated synthesis. Global deprotection afforded the target hexamer kojibiosyl-glycerol containing TA (1).     

Enzymatic synthesis of 1,3-dihydroxyphenylacetoyl-sn-glycerol: Optimization by response surface methodology and evaluation of its antioxidant and antibacterial activities

In this study, the enzymatic synthesis of phenylacetoyl glycerol ester was carried out as a response to the increasing consumer demand for natural compounds. 1,3-dihydroxyphenylacetoyl-sn-Glycerol (1,3-di-HPA-Gly), labeled as “natural” compound with interesting biological properties, has been successfully synthesized for the first time in good yield by a direct esterification of glycerol (Gly) with p-hydroxyphenylacetic acid (p-HPA) using immobilized Candida antarctica lipase as a biocatalyst. Spectroscopic analyses of purified esters showed that the glycerol was mono- or di-esterified on the primary hydroxyl group. These compounds were evaluated for their antioxidant activity using two different tests. The glycerol di-esters (1,3-di-HPA-Gly) showed a higher antiradical capacity than that of the butyl hydroxytoluene. Furthermore, compared to the p-HPA, synthesized ester (1,3-di-HPA-Gly) exhibited the most antibacterial effect mainly against Gram + bacteria. Among synthesized esters the 1,3-di-HPA-Gly was most effective as antioxidant and antibacterial compound. These findings could be the basis for a further exploitation of the new compound, 1,3-di-HPA-Gly, as antioxidant and antibacterial active ingredient in the cosmetic and pharmaceutical fields.     

Automated high-performance liquid chromatography method for the determination of rosiglitazone in human plasma

A robust, fully automated assay procedure for the determination of rosiglitazone (I, BRL-49653) in human plasma has been developed. Plasma concentrations of I were determined using automated sequential trace enrichment of dialysates (ASTED) coupled to reversed-phase high-performance liquid chromatography. Sequential automated dialysis of human plasma samples was followed by concentration of the dialysate by trace enrichment on a C₁₈ cartridge. Drug and internal standard, SB-204882 (II) were eluted from the trace enrichment cartridge by mobile phase (0.01 M ammonium acetate, pH 8–acetonitrile, 65:35, v/v) onto the HPLC column (a Novapak C₁₈, 4 μm, 100×5 mm radial compression cartridge) protected by a Guard-Pak C₁₈ cartridge. The compounds were detected by fluorescence detection, using an excitation wavelength of 247 nm, and emission wavelength of 367 nm. The lower limit of quantitation of the method was 3 ng/ml (200 μl aliquot) with linearity demonstrated up to 100 ng/ml. Within- and between-run precision and accuracy of determination were better than 10% across the calibration range. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can be safely stored for at least 7 months at −20°C. This method has been successfully utilised to provide pharmacokinetic data throughout the clinical development of rosiglitazone.     

Effect of nutritional status on nutrient and gas utilization by the mammary gland of lactating sows

Milk synthesis being a continuous process in lactating sows, the mammary gland has to adapt its metabolism in response to extreme short-term changes in nutrient availability in the arterial bloodstream, due to the feeding pattern. The objective of the present study was to better quantify and understand these adaptations. The effect of morning refeeding after an overnight 16-h feed withdrawal was measured on the uptake of energy-supplying nutrients, amino acids (AA), and some vitamins and minerals. After farrowing, catheters were fitted in the right anterior mammary vein and in the carotid artery of six sows. Blood samples were drawn on days 7, 14, and 21 of lactation, every 30 from 60 min before the morning meal to 300 min after the morning meal. Plasma concentrations of glucose, lactate, triglycerides (TG), non-esterified fatty acids (NEFA), glycerol, α-amino nitrogen (N), vitamins B₁₂, and folates were determined on all samples. Riboflavin and AA concentrations were only measured 30 min before the meal and 120 min after the meal. Arterial and venous plasma concentrations of glucose, lactate, and α-amino N increased after the meal (P < 0.01), and concentrations of NEFA, glycerol, and TG decreased (P < 0.01). Mammary arteriovenous concentration difference increased after the meal for glucose, lactate, and α-amino N (P < 0.01), remained constant for TG, and decreased for NEFA (P < 0.01) and glycerol (P < 0.05). Arterial concentrations of all AA increased after the meal, but changes of arteriovenous difference with the meal differed among AA. Arteriovenous difference of energy (7.6 kJ/l plasma) concentration was similar in feed-deprived and fed sows, but the contribution of the various nutrients differed, and the respiratory quotient was lower (P < 0.01) before the meal (0.95) than after the meal (1.54). The relative contributions of glucose, lactate, TG, NEFA, and AA to arteriovenous difference in energy concentration were 50.2, 3.8, 25.1, 0, and 20.8% in fed and 24.6, 2.2, 24.9, 32.9, and 15.0% in feed-deprived sows, respectively. The daily mammary extraction of vitamin B₁₂, estimated from arteriovenous differences was higher than the amount of this vitamin bioavailable from the diet, probably contributing to the 50% decrease in plasma concentration between day 7 and day 21 of lactation. For both riboflavin and folates, arteriovenous differences in plasma concentrations were small or not different from zero. These results indicate that the mammary gland has a great capacity to adapt nutrient uptake very rapidly and modify its metabolism according to the nutrients available in the bloodstream.     

Allergenic glaucans from dermatophytes. II. Enzymic degradation

The action of pyruvic acid on glycerol leads principally to two isomeric, bicyclic lactones 1 and 2; this reaction is compared with that employing methyl and ethyl esters of pyruvic acid. The action of pyruvaldehyde or 2,3-butanedione on glycerol leads to bicyclic heterocycles having a secondary (5) or tertiary (6) hemiacetal group, respectively. These compounds, and likewise the derived alkoxy, acetoxy, and chloro analogs, are subject to the anomeric effect, the endo isomers being thermodynamically more stable than the exo isomers.     

Glycerols and fatty acids isolated from Micractinium sp. KSF0031

From the collected extract from King Sejong Antarctic Station, strain Micractinium sp. KSF0031, led to the isolation of one new monoacyldigalactosyl glycerol (1) and seven known compounds (2–8). Their chemical structures were established using extensive spectroscopic techniques, including 1D, 2D-NMR, and MS, and compared with the published data. To the best of our knowledge, this is the first report to investigate the secondary metabolites from genus Micractinium. The monoacyldigalactosyl glycerol in Micractinium could serve as its chemotaxonomic markers.     

A rapid method for the analysis of N tau-methylhistidine in human urine

A novel, automated method is described for the determination of N^τ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for N^τ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of N^τ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment.     

Novel androgen receptor full antagonists: Design,synthesis, and a docking study of glycerol and aminoglycerol derivatives that contain p-carborane cages

Based on the co-crystal structure of bicalutamide with a T877A-mutated androgen receptor (AR), glycerol and aminoglycerol derivatives were designed and synthesized as a novel type of carborane-containing AR modulators. The (R)-isomer of 6c, whose chirality is derived from the glycerol group, showed 20 times more potent cell inhibitory activity against LNCaP cell lines expressing T877A-mutated AR than the corresponding (S)-isomer. Docking studies of both isomers with AR suggested that (R)-6c is in closer spatial proximity to helix-12 of the AR than (S)-6c, which is the most important common motif in the secondary structure of AR for the expression of antagonistic activity.     

Large scale isolation and storage of Neurospora plasma membranes.

Functionally inverted plasma membrane vesieles isolated from the eukaryotic microorganism Neurospora crassa generate and maintain a transmembrane electrical potential via ATP hydrolysis catalyzed by a plasma membrane ATPase (G. A. Scarborough, 1976, Proc. Nat. Acad. Sci. USA73, 1485–1488). In order to facilitate investigation of the molecular mechanism of the electrogenic ATPase, and other transport systems, we have developed a method for the large scale isolation and storage of Neurospora plasma membranes in a stable form. Large quantities of open plasma membrane sheets (ghosts) are isolated by a scaled-up modification of the original method (G. A. Scarborough, 1975, J. Biol. Chem.250, 1106–1111) and stored at ?26°C in 60% glycerol ($\text{v}\text{v}$). As needed, the ghosts are washed free of glycerol and then converted to closed vesicles by a modification of the original method. With this technique, plasma membrane vesicles with normal electrogenic pump activity can be prepared daily in approximately 2.5 h.     

Dependence of Balbiani ring induction in Chironomus salivary glands on inorganic phosphate

Galactose or certain other monosaccharides, administered for several days in the culture medium to larvae of Chironomus pallidivittatus, induce a new Balbiani ring, BR6, in their salivary gland chromosomes (W. Beermann, 1973, Chromosoma, 7, 198–259). This also applies to ethanol (Beermann, personal communication) and as found here, to glycerol. Induction of BR6 has previously been found to be paralleled by the appearance of one or two giant proteins (Ic₁ and Ic₂) probably deriving from allelic genes. We record here that the induction also includes the production of a new giant RNA species similar in size to the RNA from the Balbiani rings normally present, BR1 and BR2. Administration of inorganic phosphate together with glycerol prevented the appearance of BR6, as well as of the new RNA and component Ic protein(s); by contrast chloride and sulfate at similar concentrations did not prevent these effects. Administration of inorganic phosphate several days after the inducer and its continued presence reversed the effect of induction. Glycerol caused a marked depression in the level of inorganic phosphate in the hemolymph which persisted throughout its administration; the phosphate level in the glands was, however, unaffected. Inorganic phosphate administered together with the inducer at equimolar concentrations largely prevented the decrease in phosphate levels. It is concluded that a decrease in phosphate level is required for BR6 induction by glycerol. The two other inducers, galactose and ethanol, which were studied in less detail, seem to have a similar action.     

Optimization of Folin-Ciocalteu reagent concentration in an automated Lowry protein assay

The Lowry method (G. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951, J. Biol. Chem.193, 265–275) for protein concentration measurement has been automated to permit assay of samples with concentrations from 1 to 400 μg/ml. Calibration with solutions of bovine serum albumin resulted in a nonlinear (quadratic) curve. The quantity of color developed in the assay was found to be strongly dependent on the concentration of the Folin-Ciocalteu phenol reagent. Color yield peaked sharply at a reagent concentration 40% lower than that used in the Lowry procedure. Optimization of the reagent concentration is necessary to obtain maximum sensitivity from the Lowry assay.     

Glyceroglycolipids,a novel class of platelet-activating factor antagonists from Kalimeris indica

The bioassay-guided purification of chloroform extracts of Kalimeris indica (Linn) Schultz-Bip led to the isolation of three diacylglycerols: 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoylglycerol (1), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-α-(6-sulfoquinovopyranosyl)glycerol (2), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-[α-d-galactopyranosyl(1 → 6)-O-β-d-galactopyranosyl]glycerol (3). Their structures were established on the basis of spectral and chemical methods. The glyceroglycolipids 2 and 3 exhibited significant PAF receptor binding inhibitory activities. Our studies have identified diacylglycolipids as a novel class of potent PAF antagonist.     

Skeletal Maturation in the Current Pediatric Mexican Population

Objective: The most commonly used methods for bone age (BA) reading were described in the Caucasian population decades ago. However, there are secular trends in skeletal maturation and different BA patterns between ethnic groups. Automated BA reading makes updating references easier and more precise than human reading. The objective of the present study was to present automated BA reference curves according to chronological age and gender in the Mexican population and compare the maturation tempo with that of other populations.Methods: The study included 923 healthy participants aged 5 to 18 years between 2017 and 2018. A hand radio-graph was analyzed using BoneXpert software to obtain the automated BA reading according to Greulich and Pyle (G&P) and Tanner-Whitehouse 2 (TW2) references. We constructed reference curves using the average difference between the BA and chronological age according to sex and age.Results: The G&P and TW2 automated reference curves showed that Mexican boys exhibit delays in BA during middle childhood by 0.5 to 0.7 (95% confidence interval &lsqb;CI], -0.9 to -0.2) years; however, they demonstrate an advanced BA of up to 1.1 (95% CI, 0.8 to 1.4) years at the end of puberty. Mexican girls exhibited a delay in BA by 0.3 to 0.6 (95% CI, -0.9 to -0.1) years before puberty and an advanced BA of up to 0.9 (95% CI, 0.7 to 1.2) years at the end of puberty.Conclusion: Mexican children aged <10 years exhibited a delay in skeletal maturity, followed by an advanced BA by approximately 1 year at the end of puberty. This may affect the estimation of growth potential in this population.     

Automated fluorometric determination of free and total tryptophan in plasma

We have developed a sensitive automated fluorometric method based upon the manual procedure of Denckla and Dewey [(1967) J. Lab. Clin. Med.69, 160–169] for determining both free and total tryptophan in plasma. Free tryptophan is measured in a series of increasingly diluted aliquots of a sample of plasma after tryptophan bound to albumin is removed by continuous-flow dialysis. Free and total tryptophan are then derived from Scatchard plots of the data. The method can be used for nutritional assessments, clinical investigation of behavioral disorders in which serotonin is implicated in the pathogenesis, and studies on tryptophan transport and metabolism.     

Synthesis of 2-(6-aminohexanamido)ethyl 1-thio-β-d-galactopyranoside and 1-thio-β-d-glucopyranoside,and related compounds

2-(6-Aminohexanamido)ethyl 1-thio-β-d-galactopyranoside (5) and 1-thio-β-d-glucopyranoside (9) were prepared by the following scheme: 2,3,4,6-tetra-O-acetyl-1-thio-β-d-aldopyranoses, generated from 2-S-(2,3,4,6-tetra-O-acetyl-β-d-aldopyranosyl)-2-thiopseudourea hydrobromides, were aminoethylated with ethylenimine, followed by N-acylation of the products with 6-(trifluoroacetamido)hexanoic acid (1), and O-deacylation. These reactions could be carried out consecutively without isolation of intermediates, and the products obtained after gel chromatography were de(trifluoroacetyl)ated to obtain the final products. The chain lengths of the aglycons were further extended by repeating the acylation and the de(trifluoroacetyl)ation. An analog containing glycerol in lieu of a sugar was prepared by a similar reaction-scheme.     

Chemical constituents from Ligularia purdomii (Turrill) Chittenden

The first phytochemical investigation on the roots of Ligularia purdomii led to the isolation and identification of 18 compounds, including two eremophilane sesquiterpenoids (1 and 2), three benzofuran derivatives (3–5), a triterpenoid (6), two steroids (7 and 8), nine phenolic components (9–17), and a monofatty glyceride (18). The structural elucidation of the isolated compounds was performed by spectroscopic data and comparison with the literature. Compounds (−)-syringaresinol (11), scopoletin (13), 3,5-dimethoxy-4-hydroxy-benzaldehyde (14), and glycerol monolinoleate (18) have not been recorded in Ligularia genus previously. The chemotaxonomic significance of these isolated compounds has been summarized.     

Two new antioxidant flavones from the twigs of Eriosema robustum (Fabaceae)

Phytochemical study of the ethanol extract of the twigs of Eriosema robustum, a Cameroonian medicinal plant resulted to the isolation of two new flavones, 2′,3′,5′,5,7-pentahydroxy-3,4′-dimethoxyflavone (1) and 2′,3,5′,5,7-pentahydroxy-4′-methoxyflavone (2), along with five known compounds: 6-prenylpinocembrin (3), 1-O-heptatriacontanoyl glycerol (4), β-sitosterol (5), stigmasterol (6) and 3-O-β-d-glucopyranoside of sitosterol (7). The structure of the isolated compounds were elucidated on the basis of their NMR, UV and MS data, and by comparison with those reported in the literature. The ethanol crude extract, fractions and some isolated compounds (1–4) were evaluated for their radical scavenging capacity using 2,2-diphenyl-1-picryhydrazyl (DPPH). The crude extract, fraction II, the new compounds namely robusflavones A (1) and B (2) exhibited significant antioxidant activity.     

Validation of analytical method for (E)-2-decenedioic acid quantification in honey samples

Natural honey is a highly sought-after product owing to its biological uniqueness. We developed a suitable analytical method for the quantitative analysis of (E)-2-decenedioic acid in Korean natural and sugar-fed honey samples. A total of 258 honey samples were screened using ultra-performance liquid chromatography (UPLC) for the detection and quantification of (E)-2-decenedioic acid. (E)-2-decenedioic acid was present in high concentrations (122–127 mg/kg) in sugar-fed honey but only in trace amounts (10.9–14.8 mg/kg) in natural honey. Findings indicate that the UPLC method is suitable and reliable for the quantitative evaluation of (E)-2-decenedioic acid in honey. We expect that this method will be applicable for detecting honey adulteration.