In Vivo Neurochemical Characterization of Developing Guinea Pigs and the Effect of Chronic Fetal Hypoxia

Neurochemical Research - The guinea pig is a frequently used animal model for human pregnancy complications, such as oxygen deprivation or hypoxia, which result in altered brain development. To...     

Isomerization of (3S)-2,3-dihydro-5-fluoro-L-tryptophan and of 5-fluoro-L-tryptophan catalyzed by tryptophan synthase: studies using fluorine-19 nuclear magnetic resonance and difference spectroscopy

We are exploring the active site and the mechanism of the pyridoxal phosphate dependent reactions of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of substrate analogues and of reaction intermediate analogues. Fluorine-19 nuclear magnetic resonance studies and absorption spectroscopy are used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)- and (3R)-2,3-dihydro-5-fluorotryptophan. Tryptophan synthase specifically and tightly binds the 3S diastereoisomer of both 2,3-dihydro-5-fluoro-D-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan, whereas it binds 5-fluoro-D-tryptophan more tightly than 5-fluoro-L-tryptophan. Unexpectedly, we find that the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)-2,3-dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions. Since these isomerization reactions are 10(3)-10(5) times slower than the beta-replacement and beta-elimination reactions catalyzed by tryptophan synthase, they have no biochemical significance in vivo. However, the occurrence of these slow reactions does throw some light on the nature of the active site of tryptophan synthase and its requirements for substrate binding. Our results raise the interesting question of whether tryptophan synthase itself serves a catalytic role in these slow reactions or whether the enzyme simply binds the substrate and pyridoxal phosphate stereospecifically and thus promotes the intrinsic catalytic activity of pyridoxal phosphate.     

用体外免疫的人淋巴细胞建立抗破伤风类毒素的杂交瘤克隆

我们应用破伤风类毒素体外免疫的人扁桃体细胞和人-鼠异源骨髓瘤RF 系细胞进行融合,从中筛选到一株杂交瘤细胞89112—50,并通过克隆化筛选获得了两个亚克隆,其分泌的抗体是抗原特异的,和三种抗原(OVA、TC-S、FYG)都不交叉,分泌抗体的功能也比较稳定,在培养瓶内连续扩增传代13次后,仍维持相当高的抗体分泌能力,在常规传代培养过程中所收集的培养物上清液中抗体的含量平均为69.6μg/ml。     

V-fos基因对NIH3T3细胞转化的研究

本文报道了用含v-fos基因的pFBJ-2质粒转染NIH 3 T 3细胞,获得了转化细胞株。对细胞株的研究结果表明:(1)Southern杂交检测到细胞基因组中有v-fos基因的整合;(2)点杂交测得有v-fos mRNA的表达;(3)出现一系列转化表型,包括细胞形态的改变,异常的增长速率,在软琼脂上的贴壁不依赖性生长,对低血清浓度培养液的适应性以及细胞膜表而超微结构的变化等,提示v-fos基因能使NIH 3 T 3细胞发生转化,并在体外转化过程中起决定性作用。     

Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.     

Conformation of the antifreeze glycoprotein of polar fish

High-field proton and 13C NMR spectroscopy has been used to test and refine the recent proposal, based on vacuum uv circular dichroism results, of a threefold left-handed helical conformation for antifreeze glycoprotein (AFGP). Partial assignment of the protons of the glycotripeptide repeating unit has been made by comparison with spectra of model compounds, by selective decoupling, and by measurements of nuclear Overhauser effect (nOe). At 40 degrees C, AFGP fraction 8 (Mr 2600) shows 2-Hz linewidths which broaden at lower temperature. Neither 1H nor 13C chemical shifts depend strongly on temperature, suggesting no abrupt conformational transition. The nOe between alanine alpha and beta protons vary with temperature and with field strength, from small positive enhancements at 50 degrees C and 80 MHz to large negative effects at 3 degrees C and 300 MHz, indicating a substantial change of rotational correlation time with temperature. The higher-molecular-weight fraction 1-4 shows negative nOe at all temperatures. The CD spectra of fraction 1-4 show bands characteristic of the polyproline II structure at both 3 and 50 degrees C, while those bands in fraction 8 are weaker at 50 than 3 degrees C. The 1H nOe, the 13C T1, and CD data are interpreted as indicating that AFGP fraction 8 is an extended "rod-like" conformation at low temperature which becomes a flexible coil at high temperature, while fraction 1-4 is a flexible rod with sufficient segmental mobility to eliminate any long-range order.     

Reproducibility of Brain Morphometry from Short-Term Repeat Clinical MRI Examinations: A Retrospective Study

Purpose

To assess the inter session reproducibility of automatic segmented MRI-derived measures by FreeSurfer in a group of subjects with normal-appearing MR images.

Materials and Methods

After retrospectively reviewing a brain MRI database from our institute consisting of 14,758 adults, those subjects who had repeat scans and had no history of neurodegenerative disorders were selected for morphometry analysis using FreeSurfer. A total of 34 subjects were grouped by MRI scanner model. After automatic segmentation using FreeSurfer, label-wise comparison (involving area, thickness, and volume) was performed on all segmented results. An intraclass correlation coefficient was used to estimate the agreement between sessions. Wilcoxon signed rank test was used to assess the population mean rank differences across sessions. Mean-difference analysis was used to evaluate the difference intervals across scanners. Absolute percent difference was used to estimate the reproducibility errors across the MRI models. Kruskal-Wallis test was used to determine the across-scanner effect.

Results

The agreement in segmentation results for area, volume, and thickness measurements of all segmented anatomical labels was generally higher in Signa Excite and Verio models when compared with Sonata and TrioTim models. There were significant rank differences found across sessions in some labels of different measures. Smaller difference intervals in global volume measurements were noted on images acquired by Signa Excite and Verio models. For some brain regions, significant MRI model effects were observed on certain segmentation results.

Conclusions

Short-term scan-rescan reliability of automatic brain MRI morphometry is feasible in the clinical setting. However, since repeatability of software performance is contingent on the reproducibility of the scanner performance, the scanner performance must be calibrated before conducting such studies or before using such software for retrospective reviewing.     

Young Male Patients with Atrial Fibrillation and CHA2DS2-VASc Score of 1 May Not Need Anticoagulants: A Nationwide Population-Based Study

Background

It is unclear whether oral anticoagulants are beneficial for atrial fibrillation (AF) patients with low CHA₂DS₂-VASc score. Age could be important in determining the risk of thromboembolism in low risk AF patients (CHA₂DS₂-VASc score of 1 for male or 2 for female).

Methods

The Taiwan National Health Insurance Research Database (NHIRD) was used and 27,521 AF patients with CHA₂DS₂-VASc score of 1 (male) or 2 (female) not receiving anticoagulants were acquired as the study cohort, which were classified into three age groups: 20–49, 50–64, and 65–74 years. The clinical endpoint was the occurrence of ischemic thromboembolism within one year of follow up.

Results

During the follow-up of 0.94 ± 0.19 years, 385 (2.19%) male patients experienced ischemic thromboembolism, with annual rate of 2.32%. The annual risk ranged from 1.29%, 2.43% to 2.77% for male patients aged 20–49, 50–64 and 65–74 years respectively. Of the female patients, 218 (2.20%) experienced clinical event with annual rate of 2.32%. The annual risk increased from 1.87%, 2.28% to 2.64% for female patients aged 20–49, 50–64 and 65–74 years respectively. There was no difference in risk between the male patients aged 20–49 years with CHA₂DS₂-VASc score of 1 and overall male patients with CHA₂DS₂-VASc score of 0. (P = 0.631) The female patients aged 20–49 years with CHA₂DS₂-VASc score of 2 was associated with a higher risk of thromboembolic events than overall female patients with CHA₂DS₂-VASc score of 1 (HR = 1.93; P = 0.008).

Conclusions

Age is important in determining the risk of thromboembolism in AF patients with single risk factor. In male patients <50 years old with CHA₂DS₂-VASc score of 1, the risk of ischemic thromboembolism was low. Considering the benefits and the risk of bleeding, oral anticoagulation therapy may not be favorable in these patients.