Synthesis of carbamyl and ether analogs of phosphatidylcholines

A complete synthesis of 1-O-hexadecyl-2-O-N-(heptadec-8-cis-enyl)carbamyl-sn-glycero-3-Phosphocholine, a novel analog of phosphatidylcholine, has been described. Each step is simple to perform and gives the desired products in high yield. Also, some of the intermediates formed during the synthesis have been efficiently utilized to prepare 1-O-hexadecyl-2-O-oleyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphochloine and 3-O-hexadecyl-2-oeloyl-sn-glycero-1-phosphocholine. These phosphatidylcholine (PC) analogs are useful for studying the possible role of phospholipases in the capture and lyses of liposomes in vivo.     

Calcitonin mRNA activity in young obese (fa/fa) Zucker rats

Alterations in both calcitonin (CT) secretion and plasma calcium were recently described in adult obese Zucker rats. We have investigated the CT biosynthetic activity of thyroid glands in 30-day-old obese Zucker rats (fa/fa), and their controls (Lean). Plasma calcium level was significantly increased (+0.6 mg/dl) in obese animals, but plasma phosphate was unchanged. Plasma CT levels measured by radioimmunoassay (RIA) were significantly decreased in fatty (0.50 +/- 0.03 vs 0.68 +/- 0.03 ng/ml in Leans; P less than 0.001), but thyroidal hormone content was not different between Lean and fatty rats (68.7 +/- 5.1 in Leans vs 60.5 +/- 3.6 ng/gland in fatty rats). mRNA was extracted from 10 thyroids, and translated in a rabbit reticulocyte lysate (NEN) in the presence of [35S]methionine. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A 50% decrease in translatable CT mRNA was observed in fatty rats. In basal conditions, the biosynthetic activity of C cells in obese rats correlates with the secretion rate of the hormone in the face of unchanged thyroidal CT contents.     

大鼠白蛋白mRNA3''''端顺序的分子克隆(简报)

白蛋白与甲胎蛋白基因是由同一祖先基因进化而来。在小鼠,它们是一前一后定位在同一条染色体DNA上。在个体发育和肝癌变过程中,这两基因的相互关系也呈现相反的表达水平。因此,研究甲胎蛋白基因表达的调控与白蛋白基因是不可分割的。本文简要报道我们克隆了大鼠白蛋白mRNA 3′端顺序,为研究白蛋白基因结构和表达提供了必要的探针。白蛋白mRNA是从Wistar大鼠肝脏用双抗体免疫沉淀法提取。分子克隆技术基本是按“分子克隆”实验手册进行。     

The role of triiodothyronine in the regulation of synthesis rate and translatable mRNA level of fatty acid synthetase in a preadipocyte cell line

Triiodothyronine (T₃) effects on the activity, rate of synthesis and mRNA content of the key lipogenic enzyme, fatty acid synthetase, were studied in differentiating ob₁₇ preadipocytes cloned from ob/ob mouse epididymal adipose tissue. During differentiation in the presence of insulin, a 6–10-fold increase in both fatty acid synthetase specific activity and synthesis rate were reproducibly observed and occurred concomitantly. The relative synthesis rate exhibited a progressive elevation from 0.5% at confluence to a maximum level of 2% in the presence of insulin. The rate of the enzyme degradation determined by pulse-chase experiments was similar in differentiating cells and insulin-untreated cells of the same age ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 40–42 h). Furthermore, the increase in the enzyme synthesis rate was preceded by a progressively elevating amount of mRNA encoding for this protein as detected by translation in a reticulocyte lysate cell-free system. It is thus suggested that the increment in total and neosynthesized fatty acid synthetase in essentially due to an increased enzyme synthesis, reflecting an increased relative content of its specific mRNA. T₃ included at a physiological concentration (1.5 nM) in the culture medium enhanced significantly both enzyme synthesis and its specific mRNA. The most important T₃ effect was an acceleration of both processes, a stimulation of the mRNA level being detected as early as day 3 post-confluence and maximum at day 5 when the effect on the synthetase synthesis rate and activity began to be enhanced. This suggests that T₃ would mainly affect fatty acid synthetase as a pretranslational level.     

Ultraviolet-crosslinking reveals specific affinity of eukaryotic initiation factors for Semliki Forest virus mRNA

Eukaryotic initiation factors (eIF) associate readily with ³²P-labeled Semliki Forest virus (SFV) mRNA in vitro, forming complexes which can be crosslinked by 254 nm ultraviolet irradiation. After ribonuclease digestion, the initiation factors were released and analysed by gel electrophoresis. Autoradiography revealed proteins by virtue of crosslinked ³²P-labeled mRNA fragments. eIF-4A, -4B and -4C as well as three subunits of eIF-3 could be crosslinked with SFV mRNA. None of these proteins bound to ribosomal RNAs.     

Poly (8-bromo-2′-deoxyadenylic acid): The syn/anti relationship

The synthesis of a high-molecular-weight, putatively all-syn DNA analogue, poly(8-bromo-2′-deoxyadenylic acid), is described. The syn → anti transition was shown to be both salt and temperature dependent. Conditions were found which favored ‘normal’ Watson-Crick pairing and duplex formation with poly(dT).     

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.     

Suitable conditions for characterization,identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P₅₅, P₃₂, and P₂₀. P₅₅ and P₃₂ were translated from chloroplast mRNA. P₅₅ corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP₂) carboxylase; P₃₂ is probably a chloroplast membrane protein. P₂₀, the polypeptide synthesized from cytoplasmic poly(A)⁺ RNA, is the precursor of the small subunit of RuP₂ carboxylase. The balance between P₂₀ and P₃₂, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.Abbreviations RuP₂ ribulose-1,5-bisphosphate - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid

A simple and sensitive chemical assay was developed for 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. The assay is based on the liberation of ethylene from ACC at pH 11.5 in the presence of pyridoxal phosphate, MnCl₂ and H₂O₂. This assay was used to detect ACC in extracts of tomato fruits (Lycopersicon esculentum Mill.) and to measure the activity of a soluble enzyme from tomato fruit that converted S-adenosylmethionine (SAM) to ACC. The enzyme had a K_m of 13 M for SAM, and conversion of SAM to ACC was competitively and reversibly inhibited by aminoethoxyvinylglycine (AVG), an analog of rhizobitoxine. The K_i value for AVG was 0.2 M. The level of the ACC-forming enzyme activity was positively correlated with the content of ACC and the rate of ethylene formation in wild-type tomatoes of different developmental stages. Mature fruits of the rin (non-ripening) mutant of tomato, which only produce low levels of ethylene, contained much lower levels of ACC and of the ACC-forming enzyme activity than wild-type tomato fruits of comparable age.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid - SAM S-adenosyl-L-methionine Michigan Agricultural Experiment Station No. 8876     

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apoVLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellinin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.