Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method

Mouse oocytes and embryos at various developmental stages were exposed directly to an ethylene glycol-based vitrification solution (EFS) for 2 or 5 minutes at 20 degrees C. They were then vitrified at -196 degrees C and were warmed rapidly. At the germinal vesicle stage, the proportion of morphologically normal oocytes was 36 to 39% if they had cumulus cells, whereas in cumulus-removed immature oocytes and in ovulated oocytes it was only 2 to 4%. This low survival was attributed to the harmful action of ethylene glycol. After fertilization, on the other hand, the post-warming survival rate of 1-cell zygotes, as assessed by cleavage to the 2-cell stage, increased markedly (62%). As the developmental stage proceeded, higher proportions of vitrified embryos developed to expanded blastocysts; the rates increased up to 77 and 80% in 2-cell and 4-cell embryos, respectively. For embryos at the 8-cell, morula and early blastocyst stages, the proportion of embryos developed after vitrification (90 to 95%) was not significantly different from that of the untreated embryos (95 to 100%) when the period of exposure to EFS solution was 2 minutes. As the blastocoel began to enlarge, however, survival began to decrease again, with rates of 79 and 57% in blastocysts and expanded blastocysts, respectively. After the cryopreserved 2-cell, 4-cell and 8-cell embryos as well as morulae and blastocysts were transferred to recipients, 43 to 57% of the recipients became pregnant, and 48 to 60% of these various stage embryos developed into live young.     

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non–small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6β4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.     

Genetic Identification of Staphylococcus aureus by Polymerase Chain Reaction Using Single-Base-Pair Mismatch in 16S Ribosomal RNA Gene

Staphylococcus aureus is the most predominant and important pathogen in clinical microbiology. A DNA amplification assay using the polymerase chain reaction (PCR) was designed to identify S. aureus through a single-base-pair mismatch in the sequences of staphylococcal 16S ribosomal RNA (16S rRNA) genes. It was able to detect and identify S. aureus without requiring additional analytical techniques. Twenty-eight staphylococcal and non-staphylococcal strains were tested to verify the specificity of the assay, and only S. aureus strains gave a positive reaction. It may be possible to provide immediate and exact information for the identification of S. aureus.     

Inhibitory effect of azelastine hydrochloride on synthesis and release of platelet activating factor from human alveolar macrophages

The effect of azelastine hydrochloride (azelastine) on synthesis and release of platelet activating factor (PAF) in alveolar macrophages obtained from asthmatic and non-asthmatic subjects was examined. Alveolar macrophages (AMs) were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 μM) for 15 min. PAF activity was detected by aggregation of washed guinea pig platelets. PAF activity released from alveolar macrophages (AMs) from asthmatics without preincubation of azelastine was 15.97 [2.17] (mean [SD], ng/10⁷ cells) in supernatants and 42.52 [10.16] in cell pellets. After preincubation with 10⁻⁸, 10⁻⁶, and 10⁻⁴ M of azelastine, PAF activity reduced to 10.71 [2.73] (mean [SD], ng/10⁷ cells), 7.86 [0.94], and 3.52 [0.31] in the supernatants, and 35.58 [7.37], 21.57 [4.36], and 14.77 [0.99] (n = 15) in the cell pellets, respectively.PAF activity in non-asthmatic subjects without preincubation of azelastine was 8.55 [1.16] (mean [SD], ng/10⁷ cells) in supernatants and 32.64 [3.37] in cell pellets. After preincubation with 10⁻⁸, 10⁻⁶, and 10⁻⁴ M of azelastine, PAF activity reduced to 6.68 [0.78] (mean [SD], ng/10⁷ cells), 4.47 [0.51], and 2.97 [0.36] in the supernatants, and 29.53 [3.75], 14.78 [1.95], and 6.16 [0.55] (n = 20) in the cell pellets, respectively.Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra- and extracellular PAF activity from asthmatic and non-asthmatic macrophages in the same manner.     

Effect of acute hypoglycaemia on cerebral metabolic rate in fetal sheep

Studies were carried out in 11 fetal sheep four days after surgery for insertion of catheters and electrocortical leads. After a 3 h control period an insulin infusion was given to the ewe and maintained for the next 4 h. Fetal arterial glucose fell from 0.85 +/- 0.10 to 0.57 +/- 0.06 mM (SEM) while oxygen content was unchanged (3.80 +/- 0.24 to 3.75 +/- 0.21 mM). Cerebral uptake of oxygen and glucose were determined from samples drawn simultaneously from the axillary artery and sagittal vein and cerebral blood flow (microsphere technique). There was no significant change in uptake of either oxygen or glucose by the fetal brain. We conclude that a rapid fall in fetal glucose levels with no change in oxygen content does not result in decrease in cerebral metabolism measured over a short term.     

Diurnal variation of dopamine content in the rat pineal gland

Levels of norepinephrine and dopamine in the rat pineal gland were determined by a radioenzymatic assay with modifications to separate the reaction products. Catecholamines were converted to 3-O-methylated derivatives in the presence of catechol-O-methyltransferase (EC 2.1.1.1) and S-adenosyl-L-[methyl-³H]-methionine. Following solvent extraction of the labelled normetanephrine and 3-methoxytyramine, the amines were separated by high-performance liquid chromatography. Contents of both catecholamines in the pineal gland varied with a 24-hr rhythm. The content of norepinephrine was maximal at about 6 A.M. (lights on from 8 A.M. to 8 P.M.) and declined gradually thereafter. In contrast to the level of norepinephrine, the dopamine level was highest at about 0 A.M. and fell rapidly to reach a trough after the lights were turned on. These observations suggest that the diurnal variation of norepinephrine is generated by changes in the contents of dopamine in sympathetic nerve terminals innervating the pineal.     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.