Sites of synthesis of viral proteins in avian sarcoma virus-infected chicken cells.

We determined the sites of synthesis of avian sarcoma virus-specific proteins in infected chicken cells by immunoprecipitation of the products synthesized in vitro by free and membrane-bound polyribosomes; 85% of Pr76, the precursor of the viral internal structural proteins (group-specific antigens), was synthesized on free polyribosomes, and 15% was synthesized on membrane-bound polyribosomes. Pr92, the lycosylated precursor of the viral glycoproteins (gp85 and gp35), was synthesized exclusively on membrane-bound polyribomes, which is consistent with its role as a membrane protein. When we investigated the site of synthesis of pp60src, the product of the avian sarcoma virus src gene, we found that 90% was synthesized on free polyribosomes, whereas 10% was detected on membrane-bound polyribosomes. The implications of these results with respect to the subcellular location of pp60src are discussed.     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (20-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively growing, but had no effect on postconfluent cultures.     

Effect of ammonium chloride on subcellular distribution of lysosomal enzymes in human fibroblasts

Three subcellular fractions enriched in lysosomal enzyme activities have been isolated recently from human cultured fibroblasts with Percoll gradients: the dense lysosomes (DL), light lysosomes (LL), and light membranous vesicles (LM). They were shown to have different morphological, cytochemical, biochemical, and immunological properties. We now report on the dramatic but different effects of a primary amine, NH4Cl, on these subfractions. The lysosomes, as detected with a specific ultrastructural cytochemical stain for the lysosomal enzyme, arylsulfatase A, were swollen significantly in all these fractions, increasing their volumes by 64% (DL), 53% (LL), and 95% (LM), respectively. When arylsulfatase A enzyme activity was monitored, about half of the DL content was diverted to the LL. However, when newly synthesized arylsulfatase A enzyme protein was monitored with metabolic labeling and immunoprecipitation, about 80% of the enzyme protein was depleted from both the DL and LL. In contrast, neither the enzyme activity nor the newly synthesized enzyme protein of arylsulfatase A was greatly altered in the LM fraction by the treatment. Since primary amines caused newly synthesized lysosomal enzymes to diverge from the lysosomal route to a secretory pathway, it was deduced that (i) the LM fraction corresponded to a prelysosomal compartment whose lysosomal enzyme content was not affected by the amine and was thus proximal to the point of diversion between the secretory and lysosomal pathways; (ii) the LL and DL fractions were distal to the point of diversion since both fractions were depleted of their newly synthesized lysosomal enzyme; and (iii) the sorting of newly synthesized lysosomal enzyme may be different from that of the preexisting pool of the same enzyme since the LL fraction was depleted of its newly synthesized enzyme protein while accumulating excessive enzyme activity.     

Differential rates of processing and transport of herpes simplex virus type 1 glycoproteins gB and gC.

The kinetics of processing and transport of herpes simplex virus type 1 (HSV-1) glycoproteins gB and gC was investigated. The conversion of precursor to mature forms and the appearance of the glycoproteins at the infected-cell surface at different times postinfection (p.i.) were studied. gB, synthesized at 4 h p.i., was converted to the mature form with a half-time (t1/2) of 120 min and appeared at the plasma membrane with a t1/2 of 270 min. The gB synthesized at later times p.i. (6, 8, and 10.5 h) was transported less efficiently. Less than 50% of gB synthesized at later times p.i. was processed and transported to the cell surface. gB synthesized in transfected cells was transported to the plasma membrane with kinetics similar to that for gB synthesized at early times p.i. gC was processed efficiently when synthesized at both 8 and 10.5 h p.i., with t1/2 of conversion of pgC to gC of 40 and 60 min, respectively. Approximately 90 to 95% of the gC synthesized was converted to the mature form. The gC synthesized at 8 h p.i. was also transported rapidly to the cell surface, compared with the transport of gB synthesized at the same time, with a t1/2 of 240 min. Greater than 70% of the gC synthesized at 8 h p.i. appeared at the cell surface. The gC synthesized at 10.5 h was transported less efficiently to the cells surface during a 6-h chase.     

In vitro synthesis of superoxide dismutases of rat liver

The syntheses of copper, zinc-superoxide dismutase (Cu,Zn-SOD) and manganese-superoxide dismutase (Mn-SOD) in vitro were studied. Both Cu,Zn-SOD and Mn-SOD were preferentially synthesized by free polysomes. Mn-SOD was synthesized as a large precursor (26,000 daltons), which was processed to the mature size (22,500 daltons) by in vitro incubation with a rat liver mitochondrial fraction. On the other hand, Cu,Zn-SOD was synthesized as the mature size product. It was shown that Cu,Zn-SOD and Mn-SOD synthesized in vitro represented 0.018% and 0.016% of the total translation products of free polysomes, respectively.     

Carbon quantum dots fluorescence quenching for potassium optode construction

Photophysical phenomena associated with carbon nanoparticles in combination with biocompatibility and readily functionalizable properties have attracted significant interest for sensing and imaging applications. A potassium ion optode based on the fluorescence quenching of carbon quantum dots (CQDs) was constructed. The CQDs were synthesized using a microwave method, citric acid and 2,2′‐(ethylene‐dioxy)bis(ethylamine). A quantum yield of 7.1% was calculated for the synthesized CQDs. A linear dynamic range of about one‐order of magnitude with a correlation coefficient of 0.99 was obtained. The optode was applied on real samples and a 0.60–1.60% error range was obtained relative to the ion‐selective electrode.     

Hydrolysis of lipid and protein reserves in loblolly pine seeds in relation to protein electrophoretic patterns following imbibition

The correlation between changes in seed protein electrophoretic patterns and the hydrolysis of lipid and protein reserves of loblolly pine ( Pinus taeda L.) seed was studied. Seeds were incubated at 30°C for up to 12 days following stratification, then megagametophytes and embryos were assayed for lipid and protein content after each day of imbibition. The megagametophyte of mature seed was found to contain 20% lipid and 12% storage protein on a fresh weight basis. The embryo contained 26% lipid and 15% protein. Both lipid and protein reserves were depleted constantly following imbibition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble and insoluble protein fractions showed a 60 kDa protein that was representative of crystalloid-like proteins. These crystalloid-like proteins comprised 85% of the insoluble protein storage reserves. A small number of insoluble storage proteins, including a 47 kDa protein, were distinct in that they were unaffected by 2-mercaptoethanol treatment. The soluble fractions from both tissues were labelled with [³⁵S]-methionine, and incorporation was visualized by two-dimensional electrophoresis. Proteins were found to belong to one of three categories, those synthesized constitutively (comprising the bulk of newly synthesized proteins), those synthesized during germination or those synthesized after radicle emergence. Accompanying seed reserve hydrolysis were developmental shifts in protein pattern and synthesis, suggesting the possibility that control of hydrolysis is at the level of enzyme accumulation.     

Antioxidant properties of conjugates of polyethylene glycols containing terpenophenolic fragments

A series of water-soluble conjugates has been synthesized from polyethylene glycols of various lengths and 4-bromomethyl-2,6-diisobornylphenol. The membrane protective and antioxidant activities of the synthesized products were evaluated on the model of the H₂O₂-induced hemolysis of erythrocytes. It was shown that the studied conjugates have a significant antioxidant activity, and a significant membrane protective effect was shown for the conjugates containing 0.2% and 0.8% of the 2,6-diisobornyl-4-methylenephenolic fragments.     

Enzymatic synthesis of a segment of bacteriophage Qbeta coat protein gene.

The oligoribonucleotide, A-A-A-C-U-U-U-Gp, constituting a segment of RNA bacteriophage Qbeta coat protein gene was efficiently synthesized at a milligram scale by a combination of enzymatic methods using bacteriophage T4 RNA ligase and the thermophilic polynucleotide phosphorylase. A-A-A-Cp was synthesized from A-A-A and pCp by the newly developed mononucleotide addition method using T4 RNA ligase in a yield of 83%, followed by dephosphorylation with bacterial alkaline phosphatase to obtain A-A-A-C. pU-U-U-Gp was synthesized from pU-U-U and GDP by the simultaneous action of polynucleotide phosphorylase and RNase T1 in a yield of 32%. finally, the two oligonucleotides (A-A-A-C and pU-U-U-Gp) were ligated with T4 RNA ligase and the octanucleotide, A-A-A-C-U-U-U-Gp, was obtained in a yield of 85%.     

Thialysine utilization for protein synthesis by an exponentially growing E. coli culture

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.     

Synthesis of hydantocidin and C-2-thioxo-hydantocidin

Hydantocidin, a naturally occurring strong herbicide, was synthesized in an overall yield of 35.2%, with the accompanying 1'-epi-hydantocidin in overall 9.6% yield from 2,3-O-isopropylidene-D-ribono-1,4-lactone. C-2-thioxo-hydantocidin and its spiro-epimer were also synthesized in an overall yield of 14.4% and 8.5%, respectively.     

人工合成树状串联hTERT表位肽对mDC表型和功能的影响

目的 通过对树状串联hTERT表位肽（MAP）与无肽刺激组髓样树突状细胞（mDC）的相关检测,研究MAP肽对mDC的表型和功能的影响.方法 人工固相合成四分支的MAP肽,免疫磁珠分选mDC,流式细胞技术检测相关表面分子.ElISA分别检测两组mDC培养基的IL-12p70含量,并作统计学分析.结果 人工合成MAP肽纯度高（95.26 %）,免疫磁珠分选mDC纯度为72.59 %,相比于无肽刺激组mDC,MAP肽刺激组成熟（培养第9天）时MHCⅡ类分子为83.90 %、MHCⅠ类分子为91.08 %,CD86：77.03 %,CD83：92.16 %;IL-12p70的含量也大于无肽刺激组,且有统计学差异.结论 人工合成hTERT的MAP肽能足够强的激活mDC,刺激其成熟和功能的表达,可作为mDC抗肿瘤疫苗的刺激肽结构.     

The origin of cholesterol in the mesenteric lymph of the rat

These studies were performed to quantitate the amounts of newly synthesized cholesterol secreted in the mesenteric lymph of the rat and to define the origin of this cholesterol. In control animals receiving no dietary fat, the amount of newly synthesized sterol entering the lymph increased linearly with respect to time over 24 hr. When a continuous intravenous infusion of chylomicrons was given or when the animals were prefed a diet containing 2.0% cholesterol to inhibit hepatic, but not intestinal or peripheral, cholesterol synthesis, the secretion of newly synthesized sterol in lymph was markedly suppressed, suggesting that the liver was its ultimate site of origin. When the animals were subjected to either blockade of intestinal cholesterol absorption or biliary diversion, there was a decrease in both the newly synthesized and total mass of cholesterol in lymph by approximately 60%, indicating that the majority was normally derived from the absorption of luminal (primarily biliary) sterol. In the absence of dietary cholesterol, the remainder was probably derived from plasma lipoproteins that were filtered through the intestinal capillaries into the lymph. In contrast, when lymph was collected during active fat absorption, the intestine was found to secrete sterol newly synthesized by the epithelium. Such newly synthesized cholesterol was found predominantly in the unesterified fraction and accounted for approximately 27% of the total sterol found in lymph at the end of the experiment. From these studies it was concluded that in the absence of fat absorption, sterol synthesized in the intestinal mucosa was incorporated predominantly into cell membranes and did not enter intestinal lymph to any significant degree. However, during fat absorption, a fraction of this newly synthesized sterol pool was incorporated into lipoproteins and so was delivered through the intestinal lymph to the body pools of cholesterol.     

B22-D-arginine]insulin: synthesis and biological properties

An analogue of porcine insulin which differs from the native molecule in that the amino-acid residue B22-L-arginine is replaced by its D-enantiomer has been synthesized. The [D ArgB22]B-chain was synthesized by the segment condensation method and purified as the di-S-sulfonate by ion exchange chromatoggraphy on SP-Sephadex at pH 3.5. Combination with native porcine sulfhydryl A-chain gave [DArgB22]insulin which was purified by ion exchange chromatography on SP-Sephadex at pH 4.5 with a linear NaCl gradient. The biological activity of this analogue as measured by glucose oxidation in rat epididymal adipocytes was 2%. Thymidine incorporation into DNA of human fibroblast was 16%. The immunoreactivity using antipork insulin antibody in a double antibody immunoassay was 4%. The receptor-binding affinity as measured by radioreceptor assays was 2% with cultured human fibroblasts and 1% with rat adipocytes. These results suggest that the L-configuration at B22-arginine is essential for retaining the biological, immunological and receptor-binding properties of the hormone.     

Synthesis of various phosphodiesters and phosphomonoesters with ribonuclease N.

1. 3'-Guanylyl-ethanol, 3'-guanylyl-propanol, and 3'-guanylyl-alpha-glycerol were synthesized by ribonuclease N1 [EC 3.1.4.8] using guanosine 2',3'-cyclic phosphate as a phosphate donor and various alcohols as phosphate acceptors. The yields of these phosphodiesters were 15%, 13.5%, 38.2%, respectively, with respect to phosphate donor under the optimum conditions. No phosphodiester was synthesized when 2-propanol was used as a phosphate acceptor. Thus, primary alcoholic hydroxyl groups may be regarded as the preferred phosphate acceptor. 2. 3'-Guanylyl-glucose and 3'-guanylyl-ribose were synthesized using glucose and ribose as phosphate acceptors. Under the optimum conditions, the yields of guanylyl-glucose amounted to 52.0%, while that of guanylyl-ribose was much lower. The guanylyl-glucose can be regarded as 3'-guanylyl-6-glucopyranose, based on the results of periodate oxidation. 3. Neither hydroxyamino acids (serine and threonine) nor N-acetylserinamide could be phosphorylated under the conditions used for the above phosphorylations. 4. 3'-Guanylyl-glycerol obtained as above was hydrolyzed by snake venon phosphodiesterase to produce glycerol 3-phosphate. The latter consisted of L-glycerol 3-phosphate (ca 17%) and the D-isomer (ca. 83%). Ribonuclease N1 thus catalyzes an asymmetric synthesis.     

Bilirubin production and conjugation from newly formed heme in isolated rat hepatocytes.

1. Heme synthesis from delta-aminolevulinic acid (delta-ALA) in freshly isolated rat hepatocytes was maximal at 100 microM with a rate of approx. 7 nmol being synthesized per g wet weight cells. 2. Approximately 8% of synthesized heme was converted to bilirubin and 50% of the newly synthesized bilirubin was conjugated. 3. The ratio of di to monoconjugate was approx. 2.5. Incorporation of delta-ALA into bilirubin was increased by additional delta-ALA, heme and was also doubled in cells isolated from animals treated with CoCl2. 4. Bilirubin formation was inhibited approx. 90% by in vitro treatment with heme oxygenase inhibitors zinc and tin protoporphyrin.     

Solid phase synthesis of hydrophobic difficult sequence peptides on BDDMA-PS support.

This article illustrates the successful and efficient solid phase assembly of hydrophobic difficult sequence peptides following both t-Boc and Fmoc chemistry. The peptides were synthesized on an optimized 1,4-butanediol dimethacrylate-crosslinked polystyrene support (BDDMA-PS). Four difficult sequence test peptides, VAVAG, VIVIG, QVGQVELG and VQAAIDYING, were synthesized in relatively good yield and purity without any aggregation problems. The peptides were assembled on chloromethylated and 4-hydroxymethylphenoxymethyl (HMP) BDDMA-PS resins. The peptides were fabricated using Boc amino acid 1-hydroxybenzotriazolyl and Fmoc amino acid pentafluorophenyl active esters in coupling reactions. The peptides after synthesis were cleaved from the polymeric support by exposing the peptidyl resin to 90% trifluroacetic acid/5% thioanisole/5% EDT mixture. The HPLC and MALDI TOF MS studies of the peptides revealed the high homogeneity of the synthesized peptides. Chloromethylated resin having a functional group loading of 1.14 mmol Cl/g was used for the synthesis. The yield and homogeneity of these peptides synthesized using the new support were high when compared with the conventional DVB-PS resin.     

DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts

Sodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.