Functional relationships between a reticulocyte polypeptide-chain-initiation factor (IF-MP) and the translational inhibitor involved in regulation of protein synthesis by haemin

The rate of initiation of protein synthesis in rabbit reticulocyte lysates is regulated by a translational inhibitor protein which is activated in the absence of added haemin. The effects of this inhibitor on amino acid incorporation are overcome by the protein synthesis initiation factor IF-MP which binds Met-tRNAf in a ternary complex with GTP and which can transfer this complex to small ribosomal subunits. Addition of this factor to haemin-deficient lysates prevents loss of polysomes and regenerates polysomes from 80-S single ribosomes, thus confirming an effect at the level of polypeptide initiation. The ability of the initiation factor to overcome the effects of various concentrations of the translational inhibitor suggests that the inhibitor inactivates the factor catalytically rather than stoichiometrically. In a system in vitro consisting of salt-washed 40-S ribosomal subunits, initiator Met-tRNAf and GTP, the initiation factor IF-MP transfers Met-tRNAf to the subunits in the absence of any other factor or mRNA. Equilibrium buoyant density gradient analysis in CsCl shows that formaldehyde-fixed subunits carrying Met-tRNAf bound under these conditions have a buoyant density approximately 0.02 g/cm3 lower than the bulk of salt-washed subunits, suggesting that approximately 100000 daltons of additional protein are associated with these subunits. This is in marked contrast to the amounts of protein bound to subunits incubated with Met-tRNAf and GTP in the presence of a crude ribosomal salt-wash fraction. The translational inhibitor has no effect on formation of the ternary complex IF-MP-Met-tRNAf-GTP but does impair the factor-catalysed transfer of Met-tRNAf to washed subunits. The possible mechanisms of action of the inhibitor on polypeptide chain initiation are reviewed in the light of these results.     

Sudden loss of vision--(Part II). Lesions affecting the visual pathways

Disruption of purified lymphocytic choriomeningitis (LCM) virus with Nonidet P-40 in 0.5 M KCl followed by sucrose gradient centrifugation in 0.3 M KCl led to the isolation of two viral nucleoproteins (RNPs) as well as 40S and 60S ribosomal subunits. The largest viral RNP sedimented heterogenously at 123S to 148S and was associated with 23S and 31S viral RNA. The other viral RNP sedimented at 83S and was associated with 23S viral RNA. The buoyant density in CsCl was determined to be 1.32 g/cm3 for the viral RNP. Densities of 1.52 and 1.60 g/cm3 were determined for the 40S and 60S subunits, similar to those of the BHK-21 cells subunits dissociated by 0.5 M KCl. The viral RNPs were partly sensitive to RNase.     

Concomitants of short-and long-term calcitonin therapy

When Acetabularia cliftonii chloroplast DNA (p = 1.706 g/cm3) is centrifuged in an ethidium bromide-CsCl gradient, the lower band is enriched for DNA with a buoyant density of 1.712 g/cm3 containing small covalently closed circular molecules. The minicircles measure 4.15 +/- 0.30 mum in the closed conformation and 4.35 +/- 0.20 mum in the open conformation. They are not of nuclear or bacterial origin, and appear to exist as independent entities within the chloroplast, although a mitochondrial origin cannot be completely ruled out. No 40-45 mum circles, as found in other chloroplasts, were found in either ethidium bromide-CsCl fraction. None were found in total chloroplast DNA by any of a number of methods tried. Linear molecules up to 200 mum were measured in chloroplast lysates. The main chloroplast genome may consist of very large circular molecules which are broken by even small shear forces.     

Endotoxinemia in liver cirrhosis]

Ribosomes isolated from either dry viable or non-viable pea embryonic axis tissue were equally effective in the support of polyphenylalanine synthesis in a poly(U)-directed cell-free protein-synthesising system. Ribosomes isolated from imbibed non-viable axis tissue were impaired in their ability to support polyphenylalanine synthesis in the cell-free system. RNA isolated from ribosomes and 40-S ribosomal subunits of dry or imbibed viable axis tissue was found not to be degraded, whereas the equivalent RNA species isolated from non-viable axis tissue showed an increased degree of breakdown as imbibition proceeded. Even though rRNA of imbibed non-viable axis tissue was degraded, the ribosomes and ribosomal subunits of these embryos appeared intact. In viable embryonic axis tissue the percentage of ribosomes present in the cell in the form of polysomes increased during imbibition whereas no polysomes could be detected in ribosomal preparations from dry or imbibed non-viable axis tissue. The breakdown of rRNA in ribosomal particles from non-viable axis tissue may be a contributory factor to senescence and loss of viability in Pisum arvense.     

Activities of nucleoprotein particles derived from rat liver ribosome

80-S ribosomes and 60-S subunits from rat liver were treated at increasing KC1 concentrations giving protein-deficient ribosomal particles whose components were analyzed and their activity tested. Most of the activities assayed stand treatment up to KC1 concentrations of around 0.6 M; peptidyl transferase, measured by the fragment reaction, however was 50% inhibited by 0.5 M KC1 in 60-S subunits but not in 80-S ribosomes. Three proteins, L21, L26 and L31, might be implicated in this loss of activity. 60-S subunits forming part of the 80 S ribosome are more resistant to the salt treatment and the pattern of proteins released by the treatment differs from the one obtained from free 60-S subunits, implying perhaps a change of conformation of this subunit upon association to form 80-S couples. According to their resistance to release by KC1 the proteins of the large sub-unit can be divided into three groups: (1) easily removed, including proteins: L1, L11, L17 and L25 in 80-s subunits and in addition, L5, L8, L9, L13, L20, L22, L26, L29, L31 and L32/33 in 60-S subunits; (2) proteins resistant to release by high salt concentrations in 80-S ribosomes as well as in 60-S subunits, namely proteins L3, L14, L27, L36, L40, L41, X1 and X2; (3) the rest of the proteins which are released in a more or less continuous way throughout the treatment. 5 S RNA is not released by KC1 treatment at the concentrations used. The binding sites for the antibiotics trichodermin and anisomycin are affected in a different way by the salt treatment, indicating that they are structurally different.     

Specificity studies of particulate binding sites for steroid hormones in subcellular fractions of the porcine corpus luteum

We have studied the binding of a number of radiolabelled steroids and lipophilic substances to porcine corpus luteum (CL) particulate fractions. Following preincubation of CL homogenates with radiolabelled progesterone or pregnenolone prior to fractionation on continuous sucrose density gradients, a broad peak of binding was observed associated with a particulate fraction of buoyant density 1.05-1.10 g/cm3. Progesterone content also peaked at a similar buoyant density (1.06-1.12 g/cm3). Pretreatment of luteal homogenates with digitonin perturbed the buoyant density of the progesterone-binding particulate fraction to 1.10-1.14 g/cm3 and sharpened the binding peak. Progesterone content was also perturbed to a similar extent by digitonin pretreatment, without release of the steroid. Oestrogens were also sequestered by this fraction, but steroid precursors (cholesterol, cholesterol ester), corticosteroids (cortisol, corticosterone), sterol conjugates (oestrone sulphate, pregnanediol glucuronide) and other lipophilic substances (arachidonic acid, phospholipid, prostaglandins E1, E2 and F2 alpha) were not bound. Androgens were bound weakly by fractions from control gradients but, in the presence of digitonin, significant binding could be demonstrated. Radiolabelled steroids were shown to interact directly with luteal membrane fractions, rather than interacting first with cytosolic steroid receptors which then bound to membranes. Furthermore, [3H]progesterone was not bound by porcine granulosa cell particulate fractions. These observations suggest that this fraction may be involved in sequestration or packaging of progesterone for secretion by the luteal cell.     

Effects of potassium chloride concentration on protein content and polyphenylalanine synthesizing capability of 40S ribosomal subunits from canine pancreas

Ribosomal small subunits from canine pancreas were used to survey the effects of potassium chloride in the concentration range from 0.4 to 1.25 M. When combined with 60S particles, the treated 40S subunits showed no significant change in phenylalanine incorporating activity until exposure to 0.95 M KCl. Decreases in protein content of the subunits were observed at high ionic strengths. Attempts to separate dissociated ribosomal protein and the remaining core particles treated with 1.25 M KCl by centrifugation of the salt-treated particles though a 40% sucrose cushion led to the observation that ribosomal subparticles isolated in this manner retained full phenylalanine incorporating activity, whereas centrifugation through other solutions resulted in inactive or less active particles. Experiments were performed to elucidate the mechanism by which the 40% sucrose cushion was stabilizing the high salt treated 40S subunits. Two-dimensional gel electrophoresis of the proteins of the various particles isolated in the study was performed. The active 40S particles contained 23-31 protein spots. The isolation of fully active 40S subunits with fewer proteins than previously reported should simplify elucidating their role in the function of the small subunit.     

C/C坯体密度对熔融渗硅法制备的C/C-SiC复合材料摩擦行为的影响

以密度分别为0.92,1.10和1.46 g/cm3的多孔C/C材料为坯体,采用熔融渗硅法获得密度分别为1.94,1.86和1.79 g/cm3的C/C-SiC复合材料A、B和C。将C/C-SiC复合材料与40Cr钢配副进行滑动摩擦实验,研究其摩擦磨损行为。结果表明:随载荷增加,坯体密度为1.83 g/cm3的材料B的摩擦因数较稳定,基本围绕0.60波动,波动幅度0.2。材料A的摩擦因数波动幅度为0.3,而材料C的摩擦因数呈直线下降,降幅最大达0.5。但随时间延长,在试验载荷下,材料A的摩擦因数稳定性最好,波动幅度为0.07。SEM形貌表明,低载荷下,C/C-SiC复合材料的陶瓷相磨屑易聚集在摩擦膜边缘,而高载荷下磨屑分布较均匀,但摩擦表面都较粗糙,未形成完整、致密的摩擦膜。     

Cu粉特性对热导管烧结毛细结构性能的影响

研究热导管铜粉的松装密度、粉末粒度、粉末粒径分布对铜粉烧结的毛细结构体断裂强度、孔隙率、毛细力吸水通量的影响。结果表明:在烧结温度为980℃,烧结时间60 min的条件下松装烧结所得铜热导管毛细结构体综合性能良好,当铜粉松装密度2.1 g/cm~3,粒径范围100~250μm,其中粒径150~250μm的质量分数为40%~70%时,铜粉烧结毛细结构体的断裂强度为9.11~9.67 MPa,孔隙率52.6%~53.8%,毛细力吸水通量1.30×10~(-3)~1.42×10~(-3) g/(s·mm~2)。     

Respiratory-deficient mutants of Torulopsis glabrata, a yeast with circular mitochondrial deoxyribonucleic acid of 6 mu m

Purified mitochondria from the petite positive yeast Torulopsis glabrata contain a circular deoxyribonucleic acid (DNA) with a length of 6 mum and a buoyant density of 1.686 g/cm3. This DNA is absent from ethidium bromide induced respiratory-deficient mutants.     

Rationale for total nephrectomy for suspected renal cell carcinoma

Effects of hyperthermia and cell densities on inhibitory activity of ascorbic acid on DNA synthesis in Ehrlich ascites tumor cells were studied. When cells at a low density of 5 x 10(3)/ml were treated with 75 microM ascorbic acid for 1 h, DNA synthesis was inhibited after treatment at 37 degrees C and the inhibition was significantly enhanced at 42 degrees C. At a cell density as high as 1 x 10(5)/ml, however, inhibition did not occur at 37 degrees C or 42 degrees C. In contrast, dehydroascorbic acid was inactive even at a low cell density under similar conditions. Inhibitory effects of ascorbic acid on DNA synthesis were also markedly enhanced by treatment at 40 degrees C. DNA synthesis was not inhibited in the absence of the drug. Furthermore, mice transplanted with cells treated with a combination of 75 microM ascorbic acid and hyperthermia at 42 degrees C, considerably prolonged their survival time in comparison with untreated cells. Addition of ascorbic acid to hyperthermia is suggested to be an advantageous treatment for cancer.     

Properties of a previously undescribed supercoiled filamentous virus infecting papaya in Venezuela

Supercoiled filamentous virus particles with lengths of 400 to 700 nm and 3 nm wide were isolated from leaves of Carica papaya L. plants showing a mild yellowing between the veins. The morphological properties of this virus resemble those of tenuiviruses. However, it was serologically unrelated to three of the five definitive members of this group of plant viruses and had biochemical features quite different from tenuiviruses. Therefore, the virus described here is possible an unreported new virus infecting papaya for which the name of papaya mild yellow leaf virus (PMYLV) is proposed. PMYLV was mechanically transmitted to papaya and to several Cucurbitaceae species. Virus particles sedimented as one component in sucrose density gradients, containing one molecule of ssRNA with an apparent size of 6400 nucleotides which constitutes 5% of the particle weight. The buoyant density of PMYLV was 1.26 g/cm3 in cesium chloride equilibrium gradients, and the virus coat protein consisted of a single polypeptide with mol. wt. of c. 39 kDa. Estimated virus yield in purified preparations was 2.6 g/kg leaf tissues. An antiserum was produced with a titer of 1:1500. Ultrastructural observations of PMYLV-infected leaf tissues showed crystalline aggregates of virus particles, closely associated with electron dense amorphous inclusion bodies only within xylem cells.     

The ipsilateral and contralateral connections of the fifth somatosensory area (SV) in the cat cerebral cortex

THE ipsilateral and contralateral corticocortical connections to the fifth somatosensory area (SV) in the feline cortex were determined from the location of retrogradely labelled cells following a single injection of HRP into SV. The injection was made into physiologically defined components of the body representation in SV. After injection of HRP into the face regions of SV, HRP-labelled cells were located ipsilaterally in areas 6 beta, 3b and 1-2 of the primary somatosensory (SI), in the second somatosensory (SII), third somatosensory (SIII), and fourth somatosensory (SIV) areas, along the ansate sulcus, and in areas 5a and 6a beta of the ipsilateral cortex, as well as in area 1-2 of SI and in SV of the contralateral cortex. On the other hand, after HRP had been injected into the trunk/hindlimb area, HRP-labelled cells were located in areas 3a, 1-2 of SI, in area 5, in SII, in SIII and in SIV of the ipsilateral cortex, as well as in area 1-2 of SI, and in SV of the contralateral cortex. The extent of these interconnections suggests that SV receives multiple sensory inputs and may function to integrate this information.     

Peculiarities of primary and secondary structure of phage FI-1 DNA

The composition of nitrous bases of phage FI-1 DNA was studied. As is evidenced from the values of buoyant density in CsCl (p=1,7093 g/cm(3)), melting temperature (T degrees m=86,05 degrees), spectral parameters and direct chromatographic determination, the DNA analysed contains 41,5 mole% pairs of guanine-cystosine. 5-hydroxymethylcytosine and other anomalous bases were not found. Chemical identification and jaxtposition of data of buoyant density in CsCl and Cs2SO4 (p=1,4466 g/cm(3)) and T degrees m. showed the presence of the extra-sugar component in DNA, most likely in the form of hentibiose. Spectral character of thermal denaturation of DNA in different solvents is indicative of the double helixity of its structure. DNA is characterized by enthalpies of conformational transitions "helix coil" (deltaH=12,3 kcal/g) and (deltaH=10 kcal/g) for the solvents, 1 x SSC, and 0,1 x SSC, correspondingly. The presence of extra-sugar in DNA with standard set of nitrous bases is discussed.     

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.     

Studies on native ribosomal subunits from rat liver. Purification and characterization of a ribosome dissociation factor

A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, has been identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) has been purified from high salt (0.88 M KCI) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity is measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity is recovered from most of the particles that are resolved by zonal centrifugation of the total "native subunits" obtained from the postmicrosomal fraction; the highest concentration of IF-3, however, appears to be associated with native 40S subunits. The purified dissociation factor IF-3 is composed of about ten polypeptides and the molecular weight is estimated to be between 500 000 and 700 000, on the basis of glycerol and cesium chloride gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes are dissociated by IF-3, a product is formed which is dependent on the concentration of the protein factor and has the characteristics of a 40SIF-3 complex; centrifugation of the complex on sucrose and cesium chloride gradients suggests that the complex consists of 1 equiv of each of the two components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appears to interact with 60S subunits but multiple complexes are formed, some with more than 1 IF-3 equiv per 60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it does not affect polysome-associated ribosomes engaged in protein synthesis.     

Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.     

Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ

The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.     

316L不锈钢粉末高速压制行为

采用自行设计制造的18m高落锤式高速压机,研究316L不锈钢粉末的高速压制行为.实验结果表明,冲击速度增大可有效提高生坯密度,对室温粉末进行高速压制,当冲击速度从10 m/s提高到18m/s时,生坯密度从7.18 g/cm3提高到7.61 g/cm3.而在同样冲击速度下,对160℃温粉末进行高速压制时,生坯密度从7.33 g/cm3提高到7.76 g/cm3.同时生坯强度随冲击速度的提高而升高,冲击速度从10 m/s提高到18m/s时,160℃压制的生坯强度从72.5 MPa提高到94.1 MPa,室温压制生坯强度从62.1MPa提高到89.3MPa.通过对生坯SEM照片的分析,得知高速压制过程中粉末会发生严重的塑性变形和碎裂现象,孔隙的形状也会发生改变.该文还对高速压制致密化机理进行了探讨,指出在较高的速度压制时,颗粒间的摩擦和绝热剪切作用使粉末颗粒界面的温度升高,有利于粉末颗粒的塑性变形和焊合,从而有效提高了生坯的密度.     

核反应堆用碳化硼芯块常压烧结工艺研究

以核级碳化硼粉为原料,酚醛树脂作粘结剂与助烧剂,用钢模成形方法制备出碳化硼生坯,在碳管炉中用常压烧结方法在2250℃保温40 min,制备出高温气冷堆控制棒用碳化硼环形芯块。用化学分析方法分析了原料粉与碳化硼烧结体的化学成分,用排水法对芯块的密度进行测试,研究了碳质量分数对碳化硼芯块密度的影响,用扫描电镜对生坯与芯块的微观结构进行研究,对1:1大尺寸碳化硼芯块的变形量进行统计。结果表明:碳对碳化硼具有很好的助烧作用,碳化硼的密度随碳含量的增加而增加,当碳质量分数为5%时,最高密度达到2.35 g/cm~3;碳掺量为3%时,制得的1:1碳化硼芯块的密度为2.0 g/cm~3,化学成分等各项指标均满足反应堆用核级碳化硼芯块的技术条件要求。