Spontaneous Reperfusion after In Situ Thromboembolic Stroke in Mice

Injection of thrombin into the middle cerebral artery (MCA) of mice has been proposed as a new model of thromboembolic stroke. The present study used sequential multiparametric Magnetic Resonance Imaging (MRI), including Magnetic Resonance Angiography (MRA), Diffusion-Weighted Imaging (DWI) and Perfusion-Weighted Imaging (PWI), to document MCA occlusion, PWI-DWI mismatch, and lesion development. In the first experiment, complete MCA occlusion and reproducible hypoperfusion were obtained in 85% of animals during the first hour after stroke onset. In the second experiment, 80% of animals showed partial to complete reperfusion during a three-hour follow-up. Spontaneous reperfusion thus contributed to the variability in ischemic volume in this model. The study confirmed the value of the model for evaluating new thrombolytic treatments, but calls for extended MRI follow-up at the acute stage in therapeutic studies.     

Comparative proteome analysis using amine-reactive isobaric tagging reagents coupled with 2D LC/MS/MS in 3T3-L1 adipocytes following hypoxia or normoxia

Hypoxia during the expansion of adipocytes is known to contribute both to the secretion of multiple inflammation-related adipokines as well as to obesity. We therefore investigated the nature of protein changes occurring in adipocytes during hypoxia by observation of the intracellular proteins that are expressed in 3T3-L1 adipocytes. Lysates were utilized for quantitative proteome analysis using isobaric tags for relative and absolute quantitation (iTRAQ) combined with peptide separation by multi-dimensional liquid chromatography. Antioxidants and elongation factors, as well as glycolytic enzymes were increased in hypoxic adipocytes. These changes were supported by similar changes suggested by real-time PCR. The proteins showing changes are all potential targets for revering the mechanism behind the phenomenon of induction of obese adipocytes by hypoxia. This study can therefore aid in defining the proteomic changes that occur in adipocytes in response to oxygen stress, and can further characterize adipocyte metabolism and adaptation to low oxygen conditions.     

Protein modification by phosphorylation during the process of nuclear membrane dissolution in puromycin-treated mouse oocytes.

The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.     

Inhibitory activity of phosphorylated chitooligosaccharides on the formation of calcium phosphate

Chitooligosaccharides (COSs) were successfully prepared using an ultrafiltration membrane bioreactor system, and named as COS-I (10–5 kDa), COS-II (5–3 kDa), COS-III (3–1 kDa), and COS-IV (below 1 kDa), respectively. In addition, their phosphorylated derivatives were prepared by a P₂O₅ in methanesulfonic acid solution, and inhibitory activity on the formation of insoluble calcium phosphate of phosphorylated chitooligosaccharides. Phosphorylated COS-IV exhibited the highest inhibitory activity of calcium phosphate precipitation among tested chitooligosaccharides. Its inhibitory activity, especially at the concentration more than 4 mg/ml, was similar to that of CPP, which is widely used as a calcium fortifying agent increasing calcium absorbability. Therefore, phosphorylated chitooligosaccharides may be potential inhibitors of calcium phosphate precipitation.     

Tick-Borne Rickettsial Pathogens in Ticks and Small Mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.     

One-step enzymatic synthesis of blue pigments from geniposide for fabric dyeing

In this study, we describe a one-step chemoenzymatic reaction for the production of natural blue pigments, in which the geniposide from Gardenia extracts is transformed by glycosidases to genipin. Genipin is then allowed to react with amino acids, thereby generating a natural blue pigment. The β-glycosidases, most notably isolase (a variant of β-glucanase), recombinant β-glucosidase, Cellulase T, and amylases, were shown to hydrolyze geniposide to produce the desired pigments, whereas the α-glycosidases did not. Among the 20 tested amino acids, glycine and tyrosine were associated with the highest dye production yields. The optimal molar ratio of geniposide to glycine, two reactants relevant to pigment production, was unity. The natural blue pigments produced in this study were used to dye cotton, silk, and wool. The color yields of the pigments were determined to be significantly higher than those of other natural dyes. Furthermore, the color fastness properties of these dyes were fairly good, even in the absence of mordant.