Characterization of methylation of rat liver cytosolic glutathione S-transferases by using reverse-phase h.p.l.c. and chromatofocusing.

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.     

ESIMS analysis of fucoidan preparations from Costaria costata, extracted from alga at different life-stages

Four fucoidan fractions from brown alga Costaria costata, collected at different life-stages: vegetative, May (5F2 and 5F3) and generative, July (7F1 and 7F2) collections were characterized. It was found that seaweed synthesizes different set of fucoidans - one with high fucose content and substantial percentage of hexoses and uronic acid and lower sulfate content (7F1, 5F2 and 5F3) and other - highly sulfated galactofucan (7F2). Structural features of fractions 7F2 and 5F3 were predominantly determined by mass spectrometric analysis of low-molecular-weight (LMW) oligosaccharide fragments, obtained by autohydrolysis of 7F2 and mild acid hydrolysis of 5F3 fucoidans. It was found that oligosaccharides from 7F2 fractions were mainly built up of sulfated at C-2 and/or at C-2/C-4 (1→3)-linked α-l-fucopyranose residues. d-Galactose residues, sulfated either at C-2 or C-6, were found as parts of mixed di- and trisaccharides at both termini and, probably, internal. Fucose residues in 5F3 fucoidan fragments were sulfated at C-2 and sometimes at C-4. Galactose residues were sulfated at C-4 and less frequently at C-2. Resistant to hydrolysis fraction was probably a core, built up with fucose, mannose and glucuronic acid. Presumably, oligosaccharide fragments were branches at C-4 of GlcA. They were sulfated at C-2 and sometimes at C-4 (1→3)- and/or (1→4)-linked fucooligosaccharides (sometimes terminated with (1→3)-linked galactose) and sulfated at C-4 or C-2 (1→4)- or, probably, (1→6)-linked galactooligosaccharides, probably, with own branches, formed by (1→2)-linked galactose residues. Unsulfated xylose residues were probably terminal in chains built up of fucose. It was confirmed, that monosaccharide content and structure of fucoidans of vegetative algae changed following its life stage. Generative alga in general produced highly sulfated galactofucan having lower MW along with less sulfated mannoglucuronofucan with higher MW, which was extensively synthesized by vegetative algae.     

Resistance of mice with active and maternally passive immunity against Sendai virus

Resistance of mice with active and maternally passive immunity to Sendai virus infection was investigated. Mice with active immunization were convalescent ones from intranasal infection with 10(3) TCID50 of the virus [CT], ones immunized with 4 weekly intranasal injections of about 4 X 10(3) hemagglutinating units (HAU) of formalin-inactivated virus (FV) [IN], or ones immunized with 4 weekly intraperitoneal injections of about 2 X 10(3) HAU of FV [IP]. The serum neutralizing (NT) antibody titers ranged 1 : 10 to 1 : 20 in CT and IN, and 1 : 20 to 1 : 40 in IP at 4 weeks after initial immunization. NT antibody in the lung lavage was detected only in IP in 3/5. After intranasal challenge infection with 10(6) TCID50 of the virus, little or no gross lung lesion, no virus recovery and no body weight loss were observed throughout experiment, in these 3 immunized groups, whereas, control mice showed lung lesions in 4/5 (7 days), virus recovery in 4/5 at 3 days and in 1/5 at 7 days post-challenge, and body weight loss. In additional histological study, bronchiolar epithelial methaplasia and alveolar septal thickening, which were characteristic findings in non-immunized infected mice, were not observed in mice immunized with 2 biweekly injection of about 250 HA of FV and then challenged. Maternal immunity was investigated in offsprings from convalescent dams infected with 10(3) TCID50 at mating [mCT] and from dams immunized intraperitoneally with 4 X 10(3) HAU of FV at 0, 1 and 2 weeks after mating and, 1 and 2 weeks after parturition [mIP]. Serum NT antibody was not detected in mCT, but the titers ranging 1 : 20 to 1 : 40 were detected in mIP. After intranasal challenge of mCT, mIP and offsprings from non-immune mice with 10(3) TCID50 of the virus, virus recovery on day 3 was 2/4, 1/4 and 3/3, and incidence of total lung lesions on day 7 and 12 was 11/21, 2/13 and 16/16, respectively. Body weight gain was suppressed slightly in the immune groups but markedly in the non-immune control.     

Hepatic glucose regulation and metabolism in adult sheep: effects of prenatal betamethasone

Fetal exposure to synthetic glucocorticoids in sheep results in increased fetal hepatic 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and corticosteroid-binding globulin (CBG) protein levels and insulin resistance in postnatal life. The aim was to determine whether these changes persisted to adulthood and whether alterations in mediators of hepatic glucocorticoid and glucose regulation contributed to changes in metabolism. Pregnant ewes or their fetuses received either repeated intramuscular saline (MS, FS) or betamethasone injections (0.5 mg/kg; M4, F4) at 104, 111, 118, and 124 days of gestation (dG), or a single betamethasone injection at 104 dG followed by saline at 111, 118, and 124 dG (M1, F1). Offspring were catheterized at 2 and 3 yr of age and given an intravenous glucose challenge (0.5 mg/kg). Hepatic tissue was collected at 3.5 yr. At 2 yr of age, basal plasma insulin was elevated in M4 offspring and at 3 yr of age was elevated in F4 offspring. Basal insulin-to-glucose ratio was significantly elevated in M4 offspring at 2 yr of age and elevated in M1, M4, and F4 offspring at 3 yr of age. All betamethasone treatments resulted in significant increases in hepatic glucose-6-phosphatase (G-6-Pase) activity. Hepatic glucocorticoid receptor protein levels were not altered in M1 and M4 offspring but were increased in F1 and F4 offspring. Hepatic CBG protein levels were lower in F4 but not F1 offspring and were unchanged from control in M1 and M4 offspring. Prenatal betamethasone exposure results in elevated hepatic G-6-Pase activity in adulthood and may contribute to long-term changes in metabolism.     

Effect of luteinizing hormone releasing hormone pulse characteristics on comparative luteinizing hormone and follicle stimulating hormone secretion from superfused rat anterior pituitary cell cultures

We have shown that 4 ng luteinizing hormone releasing hormone (LHRH) pulses induced significantly greater luteinizing hormone (LH) release from proestrous rat superfused anterior pituitary cells with no cycle related differences in follicle stimulating hormone (FSH). Current studies gave 8 ng LHRH in various pulse regimens to study amplitude, duration and frequency effects on LH and FSH secretion from estrous 0800, proestrous 1500 and proestrous 1900 cells. Regimen 1 gave 8 ng LHRH as a single bolus once/h; regimen 2 divided the 8 ng into 3 equal 'minipulses' given at 4 min intervals to extend duration; regimen 3 gave the 3 'minipulses' at 10 min intervals, thereby further extending duration: regimen 4 was the same as regimen 2, except that the 3 'minipulses' were given at a pulse frequency of 2 h rather than 1 h. In experiment 1, all four regimens were employed at proestrus 1900. FSH was significantly elevated by all 8 ng regimens as compared to 4 ng pulses; further, 8 ng divided into 3 equal 'minipulses' separated by 4 min at 1 and 3 h frequencies (regimens 2 and 4) resulted in FSH secretion that was significantly greater than with either a single 8 ng bolus (regimen 1) or when the 'minipulses' were separated by 10 min (regimen 3). In experiment 2, at proestrus 1500, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2; LH release was significantly suppressed at pulse 2 compared to regimen 2 accentuating divergent FSH secretion. At estrus 0800, FSH response to the second pulse of regimen 4 was significantly stimulated FSH at proestrus 1900, 1500 and estrus 0800, FSH divergence was most marked at proestrus 1500. These data indicate a potential role for hypothalamic LHRH secretory pattern in inducing divergent gonadotropin secretion in the rat.     

Construction of linear GlcNAcβ1-6Galβ1-OR type oligosaccharides by partial cleavage of GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OR sequences with jack bean β-N-acetylhexosaminidase

Radiolabelled GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (1), GlcNAc beta 1-3)GlcNAc beta 1-6)Gal beta 1-OCH3 (4), GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (7), and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (10) were cleaved partially with jack bean beta-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan 4. The saccharides 1 and 7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducing N-acetylglucosamine units, but the glycan 10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAc beta 1-6Gal, GlcNAc beta 1-6Gal beta 1-OCH3, GlcNAc beta 1-6Gal beta 1-4Glc, and, in particular, GlcNAc beta 1-6Gal beta 1-4GlcNAc.     

Free oligosaccharides with Lewis x structure expressed in the segmentation period of zebrafish embryo

We previously reported that zebrafishalpha1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta1-4(Fuc alpha1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838-845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were alpha1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Gal beta1-4)(0,1,2)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]}Man beta1-4GlcNAc, and (Gal beta1-4)(0,1)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]} Man beta1-4GlcNAc beta1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-beta-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s).     

Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers (n = 35) were ovariectomized at 36 (n = 7), 66 (n = 8), 84 (n = 12) and 108 (n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment (n = 6) or were treated with a progesterone-releasing intravaginal device (n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1-F4). Mean diameter of the F1 follicle increased (P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest (P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower (P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108h, but amounts of follicular fluid IGFBP-4 were lower (P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher (P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.     

Microbiological and clinical periodontal effects of fixed orthodontic appliances in pediatric patients

The aim of this study was to evaluate changes of microbiota in ten patients undergoing orthodontic treatment. For each patient clinical examination of gingival index (GI) and plaque index (PI) were performed at the first molars at: baseline (T1), 2 (T2), 4 (T3) and 12 weeks (T4). At the same time subgingival plaque and tongue samples were taken for the microbiological study. Clinical results showed that at T4, the mean PI score was significantly lower than T1 and the GI was markedly reduced. Microbiological results showed that at T1 and T4 facultative aerobic bacteria were prevalent, whereas anaerobic bacteria were more common at T2 and T3.     

脂多糖对人支气管上皮细胞STAT1、STAT3、STAT4、STAT6表达的影响

目的观察脂多糖对人支气管上皮细胞16HBESTAT1、STAT3、STAT4、STAT6表达的影响。方法采用普通RT—PCR检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达;Western印迹检测16HBE细胞STAT1、STAT4、STAT6的蛋白表达。分别采用不同浓度的脂多糖在不同的时间点处理16HBE细胞,采用Real—timePCR的方法检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达。结果1μg／m1的LPS处理16HBE细胞1h组、0．25μg／m1的LPS处理16HBE细胞4h组、1μg／ml的LPS处理16HBE细胞4h组STAT1、STAT4的mRNA表达较正常对照组显著增高（P〈0．01）;0．25μg／ml的LPS处理16HBE细胞2h组、1μg／ml的LPS处理16HBE细胞2h组、10μg／ml的LPS处理16HBE细胞2h组STAT1、STAT4的mRNA表达较正常对照组有所增高（P〈0．05）;1μg/ml的LPS处理16HBE细胞1h组STAT6的mRNA表达较正常对照组显著增高（P〈0．01）。所有LPS处理16HBE细胞组STAT3的mRNA表达均较正常对照组减低。结论人支气管上皮细胞表达STAT1、STAT3、STAT4、STAT6的mRNA和STAT1、STAT4、STAT6的蛋白,一定剂量的脂多糖在某些时间点分别刺激了人支气管上皮细胞STAT1、sTAT4、STAT6的mRNA表达。     

Synthesis of N-acetyllactosamine-containing oligosaccharides, galectin ligands

The following spacered oligosaccharides were synthesized: GlcNAcbetal-3Galbetal-4GlcNAcbeta-sp, GlcNAcbetal-6Galbeta1-4GlcNAcbeta -sp, GlcNAcbeta -3(GlcNAcbeta1-6)Galbeta-4GllcNAcbeta-sp, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-sp, Galbeta1-4GlcNAcbetal-6Galbetal-4GlcNAcbeta-sp, Galbeta1-4GlcNAcbeta -3(Galbeta1-4GlcNAcbeta 1-6)Galbeta1-4GlcNAcbeta-sp, GlcNAcbeta1-3(Galbeta1-4GlcNAcbetal-6)Galbeta 1-4GlcNAcbeta-sp, and Galbeta1-4GlcNAcbetal-3(GlcNAcbetal-6)Galbetal-4GlcNAcbeta-sp (sp = O(CH2)2NH2). They represent N-acetyllactosamines substituted with N-acetylgly-cosamine or N-acetyllalctosamine residue at 03, O6, or at both positions of galactose. Glycosylation was achieved by coupling with N-trichloroethoxycarbonyl-protected glucosamine bromide in the presence of silver triflate.     

Hemoglobin concentration and aerobic work capacity in women following induced erythrocythemia

The effect of induced erythrocythemia on hemoglobin concentration ([Hb]) and aerobic work capacity was determined for nine women. Cycle tests were performed at prereinfusion (T1), 2 days after a placebo infusion (T2), 2 days postreinfusion of 334 ml of red blood cells (T3), 8 days postreinfusion (T4), and 14 days postreinfusion (T5). T1 and T2 responses did not differ, negating a placebo effect. [Hb] increased from 12.7 g X dl at T1 to 14.7 g X dl at T3 and then remained constant at T4 and T5. Hematocrit increased from 38.1% at T1 to 44.9% at T3 and then remained constant at T4 and T5. Submaximal O2 uptake (VO2) and stroke volume (SV) did not change from T1 through T5. Submaximal cardiac output (Q) and heart rate (HR) decreased from T1 to T3 and then remained constant at T4 and T5. Arteriovenous O2 difference increased from T1 to T3 and then remained constant at T4 and T5. Maximal VO2 was greater at T3 (2.65 l X min-1), T4 (2.66 l X min-1), and T5 (2.60 l X min-1) than at T1 (2.41 l X min-1). Physical work capacity was greater at T3 (10,740 kg X m), T4 (10,980 kg X m), and T5 (10,380 kg X m) than at T1 (8,747 kg X m). Maximal values for Q, HR, and SV were unchanged from T1 through T5. At maximum, arteriovenous O2 difference and Hb flow rate increased from T1 to T3 and then remained constant at T4 and T5. The greater postreinfusion [Hb] improved O2 transport capacity and appeared to regulate circulatory responses.     

A time-course study on the resistance of Penaeus japonicus induced by artificial infection with white spot syndrome virus

The onset and duration of resistance in experimental survivors of Penaeus japonicus produced by an intramuscular injection with white spot syndrome virus (WSSV) were surveyed by re-challenge tests with the virus conducted at weeks 1-4 and months 1-3 post initial exposure (PIE) to the virus. Virus neutralising activity in the survivors' plasma was also examined. Plasma-treated WSSV was separated from the plasma by centrifugation and then injected into na?ve shrimp, in parallel with each re-challenge test. Re-challenge tests of the survivors conducted at weeks 1-4 PIE revealed that the resistance commenced at week 3 (relative percent survival, RPS: 39%) and almost fully developed at week 4 (RPS: 58%), because statistically significant differences in survival rates were observed between the test (previously virus exposed) and control groups at weeks 3 and 4. Re-challenge at months 1-3 PIE resulted in RPS values of 67, 54 and 6%, respectively, indicating the resistance persisted until month 2. RPS values in neutralisation tests performed at weeks 1-4 and months 1-3 PIE were -5, 14, 36, 50, 100, 38 and 6%, respectively, which coincided with the RPS values in each re-challenge test conducted in parallel. The present results demonstrated that resistance of P. japonicus against the viral pathogen developed 3 or 4 weeks after an exposure to the virus, and it persisted for another month at 24 degrees C. The resistance was paralleled by a humoral neutralising factor(s) in the plasma of shrimp.     

Temporal and spatial changes of histone 3 K4 dimethylation at the IFN-gamma gene during Th1 and Th2 cell differentiation

Covalent modification of nucleosomal histones is an important mechanism for cytokine gene regulation in Th1 and Th2 cells. In this study, we analyzed the kinetics of histone H3 K4 dimethylation (H3K4me2) of the IFN-gamma gene. Minimal levels of H3K4me2 were found in naive CD4 T cells. After 5 days of differentiation, H3K4me2 levels were elevated in both Th1 and Th2 cells at the -5.3 kb, the promoter, the intronic DNase I hypersensitive sites, and 3' distal sites including the +9.5 kb and +16 kb sites. Th1 cells maintained high levels of H3K4me2 after longer time of culture. However, in Th2 cells after 14 days, high levels of H3K4me2 were detected only at the -5.3 kb and the promoter, whereas H3K4me2 was lost at the 3' distal sites and greatly diminished at the DNase I hypersensitive sites. After 28 days, Th2 cells lose H3K4me2 at all sites. Unlike the long-term primary Th2 cells, the Th2 clone D10 showed strong H3K4me2 at the IFN-gamma gene with distinctly high levels at the 3' distal sites. CD4 T cells transgenic for Hlx or infected with T-bet-expressing retrovirus produced IFN-gamma and retained high levels of H3K4me2 even after differentiated under Th2 polarizing conditions, suggesting positive roles of these two factors in maintaining high levels of H3K4me2 at the IFN-gamma gene.     

次声暴露对大鼠脾、肝脏某些酶活性的影响

目的:观察8 Hz,130 dB次声暴露不同时间对大鼠脾、肝脏某些酶活性的影响.方法:35只SD大鼠随机分为5组,即对照组,1周,2周,3周,4周组.每天次声暴露1次,每次2 h.实验后,观察大鼠脾、肝脏组织中MAO,GSH-px,SOD活性和MDA含量的变化.结果:大鼠脾脏MAO活性1周,2周时显著增高(P＜0.01),3周下降,4周时又显著增加(P＜0.05).肝脏组织MAO活性变化不明显(P＞0.05).脾脏组织中GSH-px活性在4周时明显增高(P＜0.05),肝脏组织中GSH-px活性在1周时就有显著性增高(P＜0.05).脾脏SOD活性在1周至4周均有显著性增高(P＜0.05).肝脏组织在实验期变化不明显(P＞0.05).脾脏组织中MDA含量在3周至4周时有显著性增高(P＜0.05).肝脏组织在1至2周时有非常显著的增高(P＜0.01),在3周时下降,到4周时又显著高于对照组(P＜0.05).结论:8Hz,130 dB次声暴露,大鼠脾、肝脏组织活性氧自由基、脂质过氧化物增高,抗氧化能力降低,造成对组织的损伤.     

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.     

日光照周期对大菱鲆幼鱼摄食、消化酶活力与血清激素含量的影响

研究了4种光照周期[24L﹕0D(D1)、16L﹕8D(D2)、8L﹕16D(D3)和0L﹕24D(D4)]对体重(30.5±2.0) g大菱鲆Scophthalmus maximus L.幼鱼20h (8:00am—4:00am)内摄食、消化酶活力、血清激素含量的影响。结果显示: (1)实验鱼的摄食率随光照时间的缩短而降低; D1组实验鱼每隔8h出现摄食高峰, 其他组均在8:00am及4:00pm出现摄食高峰。(2)D1、D2和D3组在12:00am和8:00pm出现肠道蛋白酶及淀粉酶活力峰值, 脂肪酶活力显著高于D4组(P<0.05)。(3)各组8:00am至8:00pm生长激素(GH)含量无显著变化, D1组4:00am时显著高于其他组(P<0.05), D4组0:00am及4:00am显著低于其他组(P<0.05); D1、D2和D3组初次摄食8h内皮质醇(COR)含量无显著变化, 8h后先升高后降低, D4组COR含量先升高后降低, 8:00pm时达到最高; D1和D2组0:00am时去甲肾上腺素(NE)含量显著高于其他组(P<0.05), D4组8:00pm时显著低于其他组(P<0.05); D2组8:00am及12:00am时三碘甲状原氨酸(T3)含量显著高于其他组(P<0.05), D4组8:00pm时显著低于其他组, 0:00am时显著高于其他组(P<0.05), D2和D3组4:00am显著低于D1和D4组(P<0.05), 各组T3最高值均出现在8:00pm。在实验条件下, 光照周期影响了大菱鲆幼鱼摄食、消化酶活力及血清激素含量。在此光照强度下, 大菱鲆养殖中以8—16h光照周期、日投喂2次为宜。     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.     

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

术中不同速率输注右美托咪定在全凭静脉麻醉中的量效关系研究

目的：以脑电双频指数（bispectral index,BIS）作为麻醉镇静程度指标,探讨不同速率输注右美托咪定（dexmedetomidine,DEX）对全凭静脉麻醉中丙泊酚用量,术中重要时点血液动力学及麻醉恢复质量的影响。方法：选择拟于全麻下行妇科腹腔镜手术的患者60例（ASAI～II级）,根据DEX输注速率不同随机分为四组,即D1、D2、D3和D4组,每组15例,麻醉诱导前四组均给予负荷剂量DEX0．5μg·kg-1,10min输注完毕,继而四组分别以0．2、0．4、0．6和0．8μg·kg^-1·h^-1输注速度持续输注至冲洗腹腔。四组麻醉诱导方法相同,术中以BIS作为麻醉深度指标,根据BIS值调节丙泊酚血浆靶浓度维持麻醉。记录入室用药前（T0）、DEX负荷量输注后（T1）、气腹即刻（T2）、气腹后5min（T3）、气腹后30min（T4）、解除气腹后5min（T5）、拔喉罩即刻（T6）、拔喉罩后1min（T7）时收缩压（SBP）、舒张压（DBP）、心率（HR）、丙泊酚平均用量、苏醒时间、拔喉罩时间、拔喉罩后15rainOAA／s评分、术中及术后24小时内不良反应的发生情况。结果：①D2、D3、D4组丙泊酚平均用量较D1组明显减少（P〈0．05）,D3、D4组丙泊酚平均用量较D：组明显减少（P〈0．05）。D3、D4组间差异无统计学意义（P〉0．05）。②与T0比较,Tl～T2时四组SBP、DBP、HR降低（P〈0．05）,T3～T4时D3、D4组SBP、DBP、HR降低（P〈0．05）,D1、D2组SBP、DBP无明显变化（P〉0．05）,T5～T7时四组SBP、DBP、HR降低（P〈0．05）;D3、D4组在T3～T4时SBP、DBP较D1、D2组明显降低（P〈O．05）,D1、D2两组间差异无统计学意义（P〉0．05）,D3、D4两组间差异无统计学意义（P〉0．05）。③D4组苏醒时间、拔喉罩时间、较Dlq组明显延长（P〈0．05）,D4组OAA／s评分较D1-3组明显降低（P〈0．05）。④D4组使用阿托品次数较D1-3组明显增多（P〈0．05）,四组术中使用麻黄碱次数和术后24小时内恶心、呕吐、寒战差异无统计学差异（P〉0．05）。结论：在妇科腹腔镜手术中,DEX作为全身麻醉辅助用药,负荷剂量0．5μg·kg-1,术中持续输注速率0．4μg·kg-1·h-1可以有效降低丙泊酚用量,使围手术期的血流动力学保持平稳,不延长苏醒时间和拔喉罩时间,且不良反应更少,值得临床推广应用。