Carbon dioxide fixation by mouse embryos prior to implantation

Mouse embryos in the stage of development prior to implantation were cultured in vitro in a medium that contained radioactive bicarbonate. The radioactivity was incorporated into the proteins and nucleic acids that were acid soluble. Uptake of radioactivity occurred into protein in the unfertilized ovum and was highest in all fractions in the early blastocyst stage. No incorporation was detected in the lipid fraction.     

Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface

Foreign DNA fragments can be inserted into filamentous phage gene III to create a fusion protein with the foreign sequence in the middle. The fusion protein is incorporated into the virion, which retains infectivity and displays the foreign amino acids in immunologically accessible form. These "fusion phage" can be enriched more than 1000-fold over ordinary phage by affinity for antibody directed against the foreign sequence. Fusion phage may provide a simple way of cloning a gene when an antibody against the product of that gene is available.     

A general method for site-specific incorporation of unnatural amino acids into proteins

A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme beta-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.     

An expanded eukaryotic genetic code

We describe a general and rapid route for the addition of unnatural amino acids to the genetic code of Saccharomyces cerevisiae. Five amino acids have been incorporated into proteins efficiently and with high fidelity in response to the nonsense codon TAG. The side chains of these amino acids contain a keto group, which can be uniquely modified in vitro and in vivo with a wide range of chemical probes and reagents; a heavy atom-containing amino acid for structural studies; and photocrosslinkers for cellular studies of protein interactions. This methodology not only removes the constraints imposed by the genetic code on our ability to manipulate protein structure and function in yeast, it provides a gateway to the systematic expansion of the genetic codes of multicellular eukaryotes.     

Evidence that glucose "marks" beta cells resulting in preferential release of newly synthesized insulin

Studies of isolated islets labeled with radioactive leucine show that glucose at a critical time "marks" islets in such a way as to cause preferential release of newly synthesized insulin. The preferential release of insulin from marked islets is relatively independent of subsequent secretagogues or rates of insulin secretion. Previous kinetic studies have indicated that the critical time at which marking occurs is after proinsulin biosynthesis but before the secretory event. Thus, secretory cells may regulate the diversion of newly synthesized material for immediate release as it is approaching or transiting the Golgi apparatus.     

Ras p21 as a potential mediator of insulin action in Xenopus oocytes

The oncogene protein product (p21) of the ras gene has been implicated in mediating the effects of a variety of growth factors and hormones. Microinjection of monoclonal antibody 6B7, which is directed against a synthetic peptide corresponding to a highly conserved region of p21 (amino acids 29 to 44) required for p21 function, specifically inhibited Xenopus oocyte maturation induced by incubation with insulin. The inhibition was dose-dependent and specific since (i) the same antibody had no effect on progesterone-induced maturation, (ii) immunoprecipitation and Western blotting indicated that the antibody recognized a single protein of molecular weight 21,000 in oocyte extracts, and (iii) inhibition was not observed with identical concentrations of normal immunoglobulin. Thus, p21 appears to be involved in mediating insulin-induced maturation of Xenopus oocytes. Furthermore, the mechanism may involve phosphorylation of p21, as p21 was found to be a substrate of the insulin receptor kinase.     

Major urinary protein complex of normal mice: origin

Mouse serum contains protein having the same charge density and molecular size as the major urinary protein complex of mice. Mouse liver (but not eight other tissues examined) incorporated amino acids labeled with carbon-14 into the complex in vitro. The degree of incorporation was greater in livers from males than from females, and was internmediate in livers from females treated with testosterone.     

Visual pigment renewal in the mature frog retina

It has been demonstrated by autoradiography that radioactive amino acids serve as precursors for proteins which are subsequently incorporated into retinal rod outer segment discs in mature animals. By the isolation and purification of visual pigment from retinas of adult frogs after injection of tritiated leucine and phenylalanine, it has been shown that at least part of this labeled protein consists of visual pigment (rhodopsin).     

Brain serotonin content: physiological regulation by plasma neutral amino acids

When plasma tryptophan is elevated by the injection of tryptophan or insulin, or by the consumption of carbohydrates, brain tryptophan and serotonin also rise; however, when even larger elevations of plasma tryptophan are produced by the ingestion of protein-containing diets, brain tryptophan and serotonin do not change. The main determinant of brain tryptophan and serotonin concentrations does not appear to be plasma tryptophan alone, but the ratio of this amino acid to other plasma neutral amino acids (that is, tyrosine, phenylalanine, leucine, isoleucine, and valine) that compete with it for uptake into the brain.     

An unusual form of lipid linkage to the CD45 peptide

Some protein kinases and phosphatases are myristoylated on their amino terminus, which perhaps contributes to subcellular localization or regulation. Glycoprotein CD45, a hematopoietic tyrosine phosphatase, was examined for fatty acid content. The CD45 protein incorporated [3H]myristate, but little [3H]palmitate. The label was not metabolized and reincorporated into amino acids or saccharides, as revealed by peptide maps of CD45 labeled with [3H]myristate, 14C-labeled amino acids, [35S]methionine, or 125I, and glycosidase treatments, respectively. The myristate label was resistant to mild alkaline methanolysis and was found in fatty acid and sphingosine, indicating an unusual form of lipid attachment to CD45.     

The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1

The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. We find that the Rag proteins--a family of four related small guanosine triphosphatases (GTPases)--interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a guanosine diphosphate-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.     

Location and chemical synthesis of a pre-S gene coded immunodominant epitope of hepatitis B virus

Immunodominant, disulfide-bond independent epitopes recognized by human antibodies to hepatitis B virus (HBV) are located within the 55-residue amino terminal portion (coded for by the pre-S region of HBV DNA) of minor HBV envelope components larger than the major protein constituents encoded by the S gene. A peptide having the sequence of the first 26 amino acids from the amino terminal methionine was synthesized and elicited antibodies (at dilutions of greater than or equal to 1 to 10(5) ) to the HBV envelope. These antibodies can be utilized for diagnostic tests. The immunogenicity of the peptide was substantially increased by covalent attachment to liposomes. The disulfide bond-independent determinants on sequences coded for by the pre-S gene may be more easily mimicked by peptide analogs than "conformational" determinants on the S-gene product.     

De novo design, expression, and characterization of Felix: a four-helix bundle protein of native-like sequence

The protein Felix was designed de novo to fold into an antiparallel four-helix bundle of specific topology. Its sequence of 79 amino acid residues is not homologous to any known protein sequence, but is "native-like" in that it is nonrepetitive and contains 19 of the 20 naturally occurring amino acids. Felix has been expressed from a synthetic gene cloned in Escherichia coli, and the protein has been purified to homogeneity. Physical characterization of the purified protein indicates that Felix (i) is monomeric in solution, (ii) is predominantly alpha-helical, (iii) contains a designed intramolecular disulfide bond linking the first and fourth helices, and (iv) buries its single tryptophan in an apolar environment and probably in close proximity with the disulfide bond. These physical properties rule out several alternative structures and indicate that Felix indeed folds into approximately the designed three-dimensional structure.     

Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets

Although the source of embryonic stem (ES) cells presents ethical concerns, their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas, insulin is produced and secreted by specialized structures, islets of Langerhans. Diabetes, which affects 16 million people in the United States, results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice, the insulin-producing cells undergo rapid vascularization and maintain a clustered, islet-like organization.     

Proparathyroid hormone: biosynthesis by human parathyroid adenomas

Biosynthesis of a precursor (proparathyroid hormone) to human parathyroid hormone was demonstrated during incubation of tissue from parathyroid adenomas. The proparathyroid hormone is labeled more rapidly than parathyroid hormone during incubation with amino acids labeled with carbon-14 and is progressively converted to the hormone. Apparent differences in the relative rate of conversion of precursor to hormone found in different tumors suggest that proparathyroid hormone may accumulate in some of the tumors and be secreted into the circulation.     

Antigens in Insulin Determinants of Specificity of Porcine Insulin in Man

Porcine insulin, which is distinguished from human insulin only in the amino acid at the C terminal of the B chain, is antigenic in man. Even if the last amino acid or the last eight amino acids are removed from the C terminus of the B chain of insulin, the altered insulin still reacts with human antibodies to porcine insulin; thus, the antigenic determinant of porcine insulin is located in a part of the molecule where the amino acid sequence is the same as it is in the corresponding part of the human insulin molecule.     

Transneuronal transfer of radioactivity in the central nervous system

After injection of tritiated amino acid into the mouse eye, radioactivity appeared in the contralateral visual cortex, indicating that some material had been transferred from optic axons to lateral geniculate neurons. The radioactivity in the cortex was about 2 percent of that arriving in the geniculate, and most of it was contained in material that appeared to be protein.     

饲料中添加抗菌脂肽对瓦氏黄颡鱼肌肉营养的影响

在基础配合饲料中添加0.5%、1%、2%、4%抗菌脂肽,饲养瓦氏黄颡鱼8周,对其肌肉一般营养成分、氨基酸营养、脂肪酸营养进行分析比较。结果表明抗菌脂肽对瓦氏黄颡鱼肌肉蛋白质、灰分含量无显著影响(P0.05)。瓦氏黄颡鱼是氨基酸组成均衡性较好(EAAI75、EAA/NEAA79%)的优质水产品,富含Glu、Asp、Arg等有助于术后病人康复的氨基酸。Lys含量较为富余,适当摄入可平衡谷物中Lys短缺,第一限制性氨基酸为Met+Cys。4%范围内添加抗菌脂肽对瓦氏黄颡鱼氨基酸组成无显著性影响(P0.05),EAAI随着AMI添加量增加逐渐降低,但仍高于75%。瓦氏黄颡鱼肌肉脂肪酸主要组成种类不少于11种,其中饱和脂肪酸3种、单不饱和脂肪酸4种、多不饱和脂肪酸4种,不饱和脂肪酸相对含量均较高,富含多不饱和脂肪酸(二十碳四烯酸、EPA、DHA)。在4%添加范围内,抗菌脂肽对瓦氏黄颡鱼肌肉脂肪酸组成无明显影响。     

褐藻酸寡糖诱导下大豆营养成分的变化

【目的】探索褐藻酸寡糖诱导大豆抗毒素生成和积累过程中,特别是当大豆抗毒素累积量达到最大时,大豆的异黄酮类化合物、氨基酸、寡糖、脂肪酸等营养成分的变化,为大豆的充分开发和利用提供科学依据。【方法】采用4%（w/v）的褐藻酸寡糖溶液作为诱导剂对大豆进行诱导。分别提取不同培养时间（0-6 d）下大豆中的异黄酮化合物、氨基酸、寡糖、脂肪酸等营养成分;利用高效液相色谱（HPLC）、全自动氨基酸分析仪、气相色谱（GC）等方法检测各培养时间下大豆营养成分的含量。【结果】经褐藻酸寡糖诱导后,大豆中的异黄酮化合物、氨基酸、寡糖、脂肪酸等含量均发生了变化。异黄酮类化合物中,大豆抗毒素的累积量在培养第5天时达到最高,由诱导前的0.01 mg·g^-1升高至1.72 mg·g^-1,第5天时香豆雌酚含量由开始时的15.74 μg·g^-1升高至664.8 μg·g^-1,染料木素含量由开始时的1.58 μg·g^-1升高至24.03 μg·g^-1,大豆苷元则由培养开始时的54.56 μg·g^-1降低至19.02 μg·g^-1。诱导组大豆中的总氨基酸含量由开始时的39.38%升高至43.45%,且苏氨酸、亮氨酸等必需氨基酸含量也有所升高,非诱导组大豆中的氨基酸含量也有一定程度的提高,但含量总体低于诱导组大豆。诱导组大豆中蔗糖含量由培养开始时的53.72 mg·g^-1减少至21.5 mg·g^-1,棉籽糖和水苏糖分别在培养第3天和第4天即被完全消耗;非诱导组大豆中蔗糖含量由培养开始时的53.72 mg·g^-1减少至23.09 mg·g^-1,且仍有少量的棉籽糖和水苏糖被检出。诱导组大豆中的脂肪酸总含量由培养开始时的14.27%降低至14.01%,但其中亚油酸的含量和比例有所增加。【结论】褐藻酸寡糖在诱导大豆获得最高大豆抗毒素含量时,增加了大豆中异黄酮类化合物含量,提高了大豆蛋白质的营养价值,消除了大豆中的胀气因子,并且在一定程度上提高了大豆的油脂品质。     

Structural basis of Wnt recognition by Frizzled

Wnts are lipid-modified morphogens that play critical roles in development principally through engagement of Frizzled receptors. The 3.25 angstrom structure of Xenopus Wnt8 (XWnt8) in complex with mouse Frizzled-8 (Fz8) cysteine-rich domain (CRD) reveals an unusual two-domain Wnt structure, not obviously related to known protein folds, resembling a "hand" with "thumb" and "index" fingers extended to grasp the Fz8-CRD at two distinct binding sites. One site is dominated by a palmitoleic acid lipid group projecting from serine 187 at the tip of Wnt's thumb into a deep groove in the Fz8-CRD. In the second binding site, the conserved tip of Wnt's "index finger" forms hydrophobic amino acid contacts with a depression on the opposite side of the Fz8-CRD. The conservation of amino acids in both interfaces appears to facilitate ligand-receptor cross-reactivity, which has important implications for understanding Wnt's functional pleiotropy and for developing Wnt-based drugs for cancer and regenerative medicine.