Cell-free translation of the vitamin K-dependent bone protein osteocalcin

The primary gene product of the vitamin K-dependent bone matrix protein, osteocalcin, has been identified by immunoprecipitation of cell-free translated proteins from 4 week rat calvariae mRNA preparations. Peptides of 9.8kd and 12kd, precipitated with a polyclonal affinity selected species specific antibody raised to purified rat osteocalcin, accounted for 1-2% of labelled proteins and were displaced by rat osteocalcin. These studies demonstrate that the 5800 molecular weight osteocalcin is synthesized as a precursor of approximately twice its size. The size of the propeptide, with a molecular weight of 4.3kd, is consistent with other known secreted vitamin K-dependent blood proteins.     

Heparin induces specific protein release from human intestinal smooth muscle cells

Human intestinal smooth muscle cells have recently been identified as the major cell type responsible for stricture formation in Crohn's disease. Heparin, a sulfated glycosaminoglycan, has been shown to be a key modulator of vascular smooth muscle cell growth both in vivo and in vitro and to affect the release of proteins from these cells. Heparin has also been shown to affect the growth of human intestinal smooth muscle cells. In this report we demonstrate that heparin, in addition to its effects on proliferation, also has very specific effects on proteins released by these cells in vitro. Examination of the culture medium proteins of heparin-treated human intestinal cells revealed an increase in three proteins of molecular weight between 150-250 kd, an increase in a 37 kd protein and a decrease in synthesis of lower molecular weight (less than 20 kd) proteins. In substrate-attached material a transient effect on a 48 kd protein was observed. No effects on intracellular labeled proteins could be demonstrated. The 35S-methionine labeled protein profile of human intestinal smooth muscle cells exposed to heparin is similar to that observed in rat vascular smooth muscle cells yet distinct differences do exist. Extracellular processing does not account for the released proteins nor is de novo protein synthesis required suggesting that altered intracellular protein processing is the mechanism for the heparin-induced protein pattern. The release of specific proteins following exposure to heparin may reflect a significant influence of this glycosaminoglycan on the metabolism of smooth muscle cells in general and particularly in the human intestine.     

A glycosylated protoxin in killer yeast: models for its structure and maturation

The type 1 killer phenotype in S. cerevisiae, mediated by secretion of an 11.5 kilodalton (kd) protein toxin, is cytoplasmically determined by the 1.9 kb M1-dsRNA plasmid. Maintenance of M1-dsRNA is dependent on the 4.5 kb L1-dsRNA because L1 encodes the capsid protein of the virus-like particles that separately encapsidate both dsRNA species. We have shown that in vitro translation of denatured M1-dsRNA produces M1-P1, a 32 kd protein containing the toxin peptides. We now demonstrate the presence of an unstable, 42 kd, membrane-associated, glycosylated protoxin in killer cells, probably derived from M1-P1 by cotranslational processing, and glycosylation. In vitro cotranslational processing of M1-P1, derived both from in vivo mRNAs and from denatured M1-dsRNA, produces a product resembling protoxin. Processing involves loss of 1.6 kd of protein, presumably an N-terminal leader peptide, and glycosylation. This information, together with data on in vitro expression of suppressive deletion mutants of M1-dsRNA, allows construction of testable models for the functional sequence of M1-P1 and for its maturation to toxin.     

Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non-toxic mutant proteins synthesized in vitro. Proteins obtained by N-terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N-terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C-terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment-derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N-terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease-inaccessible polypeptide of 18 kd (P18) was observed.     

The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.

The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.     

Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia

Synthesis of the low density lipoprotein (LDL) receptor was studied by incubation of cultured human fibroblasts with 35S-methionine followed by immunoprecipitation with a monoclonal antireceptor antibody. The receptor was synthesized as a precursor with an apparent molecular weight of 120 kilodaltons (kd) that was converted to a mature form of 160 kd. This novel form of processing occurred 15-30 min after synthesis and did not appear to be due to the simple addition of N-linked oligosaccharide chains. Fibroblasts from a child with the phenotype of homozygous familial hypercholesterolemia showed a disruption in receptor processing. This child has two different mutant alleles at the LDL receptor locus. One allele, inherited from his heterozygous mother, produces an abnormal 120 kd protein that cannot be processed to the mature 160 kd form. The other allele, inherited from his heterozygous father, produces a receptor that is synthesized as an elongated 170 kd precursor which undergoes a 40 kd increase in molecular weight to form an abnormally large receptor of 210 kd.     

Purification and characterization of the D2 cell adhesion protein: Analysis of the postnatally regulated polymorphic forms and their cellular distribution

The developmentally regulated, D2 cell adhesion protein has been purified from 10–12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5–1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (>200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.     

Demonstration of a boar testicular protein band that is immunoreactive to proacrosin and proacrosin binding protein antibodies.

A testicular protein band has been identified and shown to be immunoreactive to both of the proacrosin (53-55 kd) and the proacrosin binding protein (28 kd) antibodies. pH 4.5 extracts of boar testis were prepared and subjected to Western blot analysis using polyclonal antibodies of the proacrosin and the proacrosin binding protein. In addition to their respective antigens, a distinct high molecular weight protein band of approximately 200 kd was detected by both of the antibodies. Gelatin SDS-PAGE analysis of the extracts showed that this protein band was proteinase active. These results suggest that the proacrosin molecule is present as a much higher molecular weight form in the boar testis than the currently known 53-55 kd forms that have been isolated from spermatozoa.     

Novel nontoxic heat shock protein 90 inhibitors having selective antiproliferative effect

Almost all heat shock protein 90 inhibitors reported so far, which are natural product derivatives, have problems mainly with toxic side effects, and with bioavailability and solubility. In our earlier studies, we compared the steric conformational structures of substance P[6-11] with our substance P antagonists in silico, and used the diverse biological effects of these compounds as tools in our modeling and design studies for discovering antiproliferative drugs. Here, we present a new synthesized short peptide-derivative compound family that inhibits only the function of the tumor cell's heat shock protein 90 and selectively kills in vitro more cancer cells than normal cells. During the lead generation, we observed that the difference between the most effective inhibitors was only one residue or group that caused diverse effects in vitro on the studied cell lines. According to our in vivo experiments on nude mice bearing lung cancer xenografts, the inhibitors restrained tumor growth, but not caused overt toxicity. We undertook NMR spectroscopy studies to analyze the 3D molecular structural differences of our inhibitors that control their binding to the target molecule. In conclusion, we demonstrated the efficacy of new selective and small molecule anticancerogen heat shock protein 90 inhibitors with peptide nature, without in vivo toxicity on nude mouse xenograft model. Our results also shed light on the mechanism of anticancerogen action of some substance P antagonists and their derivatives.     

Biosynthesis of a renin binding protein

The biosynthesis of a porcine renin binding protein (RnBP), which specifically binds to renin and forms an inactive high molecular weight renin, was investigated. mRNAs from various porcine tissues were used to investigate in vitro protein synthesis. The kidney mRNA directed the synthesis of a high level of RnBP, whereas the liver, adrenal and pituitary gland mRNAs gave as low but significant level of it. The in vitro synthesized RnBP as well as the immunologically detected RnBP synthesized in vivo had the same molecular weight, 42,000, as that of the purified protein. Moreover, both the human and rat kidney mRNAs directed the synthesis of this protein identified with an anti-porcine RnBP antibody. These results strongly indicate that RnBP, present in various mammalian species, is synthesized in renin-producing tissues as the mature size and undergoes binding with renin without proteolytic processing.     

Vitamin D-dependent calcium-binding protein. Immunochemical studies and synthesis by placental tissue in vitro

A protein similar to rat intestinal calcium-binding protein (CaBP) has been identified in both mouse placenta and mouse small intestine. The mouse protein had a molecular weight of approximately 10,000, exhibited cation-binding properties, and demonstrated immunologic identity with vitamin D-dependent rat CaBP. Under normal dietary conditions, the concentrations of CaBP in mouse placenta and intestine increased 6- and 3-fold, respectively, during the third trimester of pregnancy in parallel with the fetal demands for skeletal mineral. Studies of in vitro protein synthesis indicated that CaBP was synthesized by placental tissue. Slices of mouse or rat placental tissue (12-18-day gestation) were incubated with [3H]leucine and the biosynthesis of placental CaBP was quantified by an immunoprecipitation method using rabbit antiserum to rat intestinal CaBP. Sodium dodecyl sulfate gel electrophoresis of the radioactive immune complex revealed a single 3H-labeled peak corresponding to the molecular weight of rat and mouse CaBP (10,050). The amount of CaBP synthesized by mouse placental tissue was dependent upon gestational age of the placenta and reflected the in vivo changes in placental CaBP content observed during gestation. These data indicate that CaBP is synthesized by placenta and provide an in vitro model for studying the developmental control of placental CaBP synthesis.     

A cytotonic, cholera toxin-like protein produced by Campylobacter jejuni.

1. Campylobacter jejuni is a major cause of gastroenteric infection. 2. This organism appears to produce both cytotonic and cytotoxic virulence factors. 3. We report here that culture filtrates of some clinical isolates of C. jejuni induce elongation of Chinese hamster ovary (CHO) cells in vitro but do not cause inhibition of fluid absorption in the rat ileum. 4. These culture filtrates contain low levels of a protein which cross-reacts immunologically with the cholera toxin. 5. The cholera toxin-like protein of C. jejuni behaved identically to cholera toxin on non-denaturing polyacrylamide gel electrophoresis. 6. Under denaturing conditions, however, this protein displayed no subunit structure and a molecular weight of approximately 50 kDa with many higher molecular weight aggregates. 7. In conclusion, isolates of C. jejuni produced small amounts of enterotoxin when grown in vitro. 8. The toxin cross-reacted immunologically with cholera toxin and has a similar native structure, but does not appear to possess subunits.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

体外合成大鼠血清淀粉样A蛋白

 血清淀粉样A蛋白(Serum Amyloid A-SAA)是一个急性期蛋白,在人和小鼠血浆中长时间维持较高的浓度,即会发展为病理学的淀粉样变。但是在大鼠中从未发现这种情况,而对大鼠SAA的研究人们还一无所知。我们使用体外系统研究了大鼠血清淀粉样A蛋白的转录和转译。在体外系统中合成的大鼠SAAmRNA的分子量约为0.5～0.56kb,用合成的mRNA转译的蛋白质分子量为8.2～10kD。用体外系统转录和转译的结果证明,这个体系重复性好,与体内的结果相似。     

Calcium-Dependent 4-aminopyridine stimulation of protein phosphorylation in squid optic lobe synaptosomes

When intact synaptosomes were incubated with [gamma-32P]ATP, maximal protein phosphorylation was attained 2 min after the start of incubation. Protein phosphorylation under basal conditions was dependent on external Ca2+, and the dominant peak of phosphorylation was a 50-kd protein. Incubation of intact synaptosomes in the presence of 3-6 mM 4-aminopyridine (4-AP) caused a markedly enhanced phosphorylation of high molecular weight proteins of 90, 100, 130, and 180 kd, with no increase in the 50 or 38 kd proteins. This effect of 4-AP was dependent on external calcium ions in the incubation medium. The 4-AP effect on the high molecular weight proteins was also found in synaptosomal plasma membranes isolated from the synaptosomes. Tetraethylammonium (TEA) ions did not produce this enhancement of phosphorylation.