Poly(A)-containing RNA in early embryogenesis of sea urchins

The content, biosynthesis and template activity of poly(A)+ RNA in the early stages of sea urchin development have been studied. The amount of poly(A)+ RNA reaches a maximum at the middle blastula stage in polyribosomes and at the 8-blastomere stage in the cytoplasm. Poly(A)+ RNA synthesis becomes noticeable at the 64-blastomere stage and the spectrum of newly synthesized molecules is different from that at the middle blastula stage. The products of translation in vitro of poly(A)+ RNA at all the stages studied show insignificant differences and contain a major group of polypeptides of molecular mass 10-20 kDa.     

Brain Actin Synthesized In Vitro Undergoes Two Different and Sequential Posttranslational Modifications

Abstract: We have studied the posttranslational processing of actin molecules synthesized in a cell-free system. The results of these experiments indicate that during the in vivo synthesis of the actins from rat brain the primary translational products undergo two different and sequential posttranslational modifications. These modifications are accompanied by slight changes in the isoelectric points of the proteins and can be detected by isoelectric focusing analysis. The same posttranslational modifications can be detected during the in vitro synthesis of chick embryo skeletal muscle actin. The evidence presented suggest that the first posttranslational modification may correspond to the methylation of a histidine residue, and the second modification most likely corresponds to the acetylation of the NH₂-terminal amino acid residues of actin molecules.     

昆明小鼠精子冷冻的研究(简报)

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受体,生长发育并产出幼仔。胚胎体外培养获得足够的晚桑葚胚和早期囊胚是胚胎干细胞建立与制备嵌合体小鼠的成功关键,暂时不用的胚胎应仍能耐受冷冻保存,待复苏移植,建立新的繁殖群。但小鼠精子冷冻研究在我国开展的十分有限,缺乏相关资料数据。本实验对不同周龄SPF级昆明(KM)小鼠进行精子冷冻及冻融精子IVF,并将IVF获得的2-细胞胚胎分别进行胚胎移植、体外培养、胚胎冷冻及复苏后移植,应用冻融小鼠精子进行胚胎工程实践。     

Differences in the Neurons That Project from the Dorsal Column Nuclei to the Diencephalon,Pretectum, and Tectum in the Cat

The dorsal column nuclei (DCN) project to a number of targets in the nervous system besides the ventroposterolateral nucleus (VPL) of the thalamus. Recent evidence obtained using double-labeling techniques indicates that DCN's diencephalic-projecting neurons differ in their location and morphology from those that project to some of its other targets, such as the cerebellum and tectum. The purpose of the present study was to characterize anatomically the DCN neurons that project another of DCN's targets, the pretectum, and to determine if any of these neurons have collateral projections to the tectum or diencephalon.

The projections were studied using two double-labeling methods. One method made use of either tritiated inactivated horseradish peroxidase ([³H]apoHRP) or tritiated N-acetyl wheatgerm agglutinin ([³H]WGA) as a marker and HRP or WGA conjugated to HRP. The other method made use of the dyes Fast Blue and Nuclear Yellow. In each cat, one marker was injected into the DCN-recipient portions of the pretectum, tectum, or diencephalon, and the other marker was injected into another of these three targets.

Neurons labeled by pretectal or tectal injections were of all sizes, fusiform and multipolar in shape, and similarly located. They were scattered through the rostral zone of DCN, but were distributed at the periphery of and at the junction between the gracile and cuneate nuclei in DCN's middle and caudal zones.

In contrast to the pretectal-and tectal-labeled neurons, neurons labeled by diencephalic injections were round and large. They were found throughout the DCN complex, but were concentrated in DCN's middle and caudal zones. When both the pretectum and diencephalon were injected in the same cat, the two groups of neurons occupied similar locations in the rostral zone, but were distinct in the middle and caudal zones, with the pretectal-projecting neurons surrounding the clusters of diencephalic-projecting neurons. Very few neurons were double-labeled.

These results demonstrate that the projections to the pretectum, tectum, and diencephalon originate from different populations of neurons within specific domains in DCN. When these results are compared with the results of electrophysiological and other anatomical studies, it appears that the pretectal- and tectal-projecting neurons may be part of a previously unrecognized system originating in DCN. In contrast with the well-known lemniscal system, recognized for its function in tactile discrimination, and composed of DCN's VPL-projecting neurons together with VPL's projections to the cerebral cortex, this other system may serve some role in the regulation of posture or the coordination of movement.     

小鼠对天花粉蛋白体内及体外免疫应答的基本特点

C 57 BL/6 J小鼠在应用弗氏全佐剂或铝矾为佐剂结合天花粉蛋白免疫后都能引起抗原特异的IgE类抗体的反应以及其它Ig类抗体的反应;IgE的滴度和总的抗天花粉蛋白抗体的滴度的动态变化趋势基本一致,但并不完全同步。IgE类上升得较IgG等类抗体为慢,但上升的速度要快。脾和肠系膜淋巴结细胞分泌抗体的动态变化也和血清并不完全一致。天花粉蛋白在一定浓度下能抑制小鼠T淋巴细胞在体外的抗原特异的增殖反应和ConA反应。这抑制并不限于增殖过程的启动。补充外源性的IL-2也不能消除这抑制作用。天花粉蛋白加热变性后丧失了对小鼠淋巴细胞的毒性,并能引起经未变性天花粉蛋白体内致敏的小鼠的T淋巴细胞在体外的明显增殖。应用微量的天花粉蛋白为抗原,以及少量的无凝集素的conA条件培液等能在体外诱发致敏的B淋巴细胞产生二次抗体应答。     

In vitro synthesis of oat globulin

Thermal denaturation of cytochrome P450 is shown to be a complex process which occurs in two steps. The first (about 50°C) takes place in several stages which can be attributed to denaturation of different regions in the cytochrome P450 with different stability. The second transition (about 90°C) is fully reversible and similar to those described for other hemoproteins.     

Generation of ribosome nascent chain complexes for structural and functional studies

Biochemical and structural studies of co-translational folding, targeting and translocation depend on an efficient methodology to prepare ribosome nascent chain complexes (RNCs). Here we present our approach for the generation of homogenous and stable RNCs involving in vitro translation and affinity purification. Fusing the SecM arrest sequence, which tightly interacts with the ribosomal tunnel, to the nascent polypeptide chain significantly enhanced the stability of the RNCs. We have been able to increase the yield of the affinity purification step by engineering a tag with higher affinity. The RNCs generated with this approach have been successfully used to obtain 3D cryo-electron microscopic reconstructions of complexes with the signal recognition particle and the translocon. The established procedure is highly efficient and if scaled up could yield milligram amounts of RNCs sufficient for crystallization experiments.     

猪卵母细胞体外成熟与冷冻附睾精子的体外受精

由屠宰母猪卵巢分离得到505枚卵母细胞,经成熟培养,其中468枚卵丘细胞扩张,成熟率为92.7％。采用冷冻的附睾精子授精,受精率为41.9％(57/136),其中单精受精率36.8％(50/136),多精受精率5.1％(7/136)。授精卵体外培养24和48小时后的卵裂率分别为5.3％(15/258)和12.9％(30/232)。把35枚早卵裂期胚胎和250枚未见卵裂的1-细胞期受精卵移入4头受体母猪的输卵管内,其中2头妊娠,分别于1990年4月10日和29日正常分娩,共生产仔猪21头。     

Aluminum and low pH effects on translatable RNA population from bean calli

Summary We have reproduced in vitro aluminium toxicity on bean calli for the purpose of analyzing how gene expression is modified by aluminum ions (Al). We have used three different media. L3m with reduced Ca and P_i concentration and a pH of 5.7; L3m4, similar, but with a pH of 4.0; and L3m4Al with the same composition and pH as L3m4, but with Al salt (AlCl₃) (500 mg/l) added. We cultured genotypically identical calli for 24 h and 1 month in the three media. Total RNA was obtained from all the calli and in vitro translated. The polypeptides obtained were resolved by two-dimensional polyacrylamide gel electrophoresis (2-D-PAGE) and compared. After the 24 h of treatment the three patterns were similar and only quantitative differences between L3m4Al-cultured and calli cultured in the two other media were detected. These differences disappeared after one month of treatment and new spots were detected in 2-D-PAGE of L3m4- and L3m4Al-cultured calli, but not in L3m. The differences observed after the 24-h treatment could be due to the ageing of the calli rather than to Al toxicity, and those described at 1 month seemed to be mostly related to pH. We suggest that Al does not specifically affect gene expression. The induced changes appeared later in time and were mainly related to low pH; only one polypeptide was associated with Al after the 1-month treatment.     

Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system

The dependence of efficiency of translation initiation on mRNA sequence parameters was investigated in an Escherichia coli in vitro expression system. We designed a large-scale expression experiment focussing on the influence of sequence variations in the translated region (TR) of the mRNA without changing the 5'-untranslated region (5'-UTR). The level of translated protein from 756 expression constructs was measured and the influence of a large number of possible effector attributes was statistically analyzed. Base exchanges immediately adjacent to the start codon up to nucleotide (+)25 had a profound effect on translational efficiency. Correlation analysis revealed a significant dependence on base pair probability and G+C content on the expression level, indicating that mRNA secondary structure in this region hampers translation. Using our training data, we developed a methodology to predict and improve the translation efficiency of open reading frames (ORFs).     

Effects of phenazine ethosulfate during culture of bovine embryos on pregnancy rate, prenatal and postnatal development

The objective was to evaluate the use of phenazine ethosulfate (PES) during culture of embryos on fetal and postnatal calf development. Oocytes collected from abbatoir-derived ovaries were matured and fertilized, and the resulting embryos were cultured in vitro by standard procedures, in a chemically defined medium plus BSA. Day 0 of culture was 18 ± 2 h after the onset of IVF. From 2.5 to 6.5 d, half of the eight-cell embryos served as controls, and the remainder were exposed to 0.3 μM PES, which decreases lipid content of embryos. Good-quality blastocysts exposed to PES (n = 38) or control (n = 35) blastocysts were transferred nonsurgically to synchronized recipients in estrus 6-7.5 d earlier, resulting in 9 calves in each group. These in vitro-produced pregnancies were evaluated weekly between 35 and 98 d postestrus by ultrasonography, and postnatal development of the calves was monitored for 1 month. Based on a limited number of transfers, use of PES during in vitro culture did not affect pregnancy rates compared to the control at 35 or 98 d (P > 0.1). Transfer at 7-7.5 d after estrus resulted in higher 98-d pregnancy rates than at 6-6.5 d (34% vs. 10%; P < 0.05). In vitro-derived fetuses that aborted had retarded fetal and placental development compared to those that went to term, but there was no difference in fetal loss between the PES treatment and controls. One calf in the PES group weighing 36 kg was born dead at 252 d of gestation, and another calf in this group was dead some hours after birth and weighed 22.2 kg when parturition was induced at 310 d of gestation. It is unclear whether these two abnormal calves were caused by the PES treatment, or were due to in vitro procedures in general. In conclusion, the use of PES during in vitro culture had no effect (P > 0.1) on pregnancy rates, conceptus losses between Days 35 and 98 of pregnancy, nor fetal postnatal development in calves born normally.     

Femtoliter compartment in liposomes for in vitro selection of proteins

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.     

川金丝猴口腔的观察

本文对川金丝猴的口腔进行观察,发现川金丝猴牙齿前后径总的趋向是Ｉ１＞Ｉ２,Ｉ１＞Ｉ２,Ｉ１＞Ｉ１,Ｉ２＞Ｉ２,上犬齿＞下犬齿,Ｐ４＞Ｐ３,Ｐ４＞Ｐ３,Ｍ２＞Ｍ３＞Ｍ１,由于Ｍ３多一下次小尖,故Ｍ３＞Ｍ２＞Ｍ１。Ｉ１与Ｉ２大小有别,但看不到Ｉ２顶端明显变尖的迹象。犬齿的性二型主要表现在上犬齿,且主要表现于与Ｐ３、Ｐ４前后径的相对大小上。雄性上犬齿与Ｉ２的间距明显大。对金丝猴的年龄判断提出以切齿更换,咬合面形态、齿星出现,齿星形态等咬合面磨损规律的口齿鉴定法。对唇游离缘近口角处明显增厚及其性差别,腭扁桃体的大体形态作了描述。此外,对舌、腭、口腔腺也作了叙述和讨论。     

Synthesis of alpha 2-macroglobulin in rat hepatocytes and in a cell-free system

The biosynthesis and secretion of alpha 2-macroglobulin was studied in rat hepatocyte primary cultures. After immunoprecipitation of alpha 2-macroglobulin from a cell homogenate and the hepatocyte medium, two forms of alpha 2-macroglobulin with app. Mr of 176000 and 182000, respectively, were identified. A precursor-product relationship for the two alpha 2-macroglobulin forms was demonstrated by a pulse-chase experiment. The cellular form of alpha 2-macroglobulin could be deglycosylated by endoglucosaminidase H, whereas the medium form of alpha 2-macroglobulin remained unaffected. On the other hand, only the medium form of alpha 2-macroglobulin was found to be susceptible to neuraminidase. In vitro translation of rat liver poly(A)+ RNA resulted in a translation product of an app. Mr of 162000.     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。