13C Nuclear magnetic resonance study of salt induced conformational change of basic polypeptides: Poly (L-lysine), poly (L-arginine), and poly (L-ornithine)

A ¹³C-nmr study of the salt-induced helix–coil transition of the basic polypeptides poly(L -lysine) [(Lys)_n], poly(L -arginine) [(Arg)_n], and poly (L -ornithine) [(Orn)_n] was performed to serve as a reference of the helical portion of histones and other proteins. As is the case with pH-induced helix–coil transition, the downfield displacement of the C^α and carbonyl carbon signals are observed in the helical state. The upfield shift of the C^β signals, on the other hand, is noted in the salt-induced transition. Regardless of the differences in the side chains and also the salts used, very similar helix-induced chemical shifts are obtained for (Lys)_n and (Arg)_n. However, the displacement of the C^α, C^β, and carbonyl carbons of (Orn)n in the presence of 4M NaClO₄ is found to be almost 50% of that of (Lys)_n and (Arg)_n. This is explained by the fact that the maximum helical content is about 50%, consistent with the ORD result. Further, the motion of the backbone and side chains of the helical from was estimated by measuring the spin-lattice relaxation time (T₁), nuclear Overhauser enhancement (NOE), and line width. In the case of (Lys)_n, the motion of the side chains is charged very little in comparison with that of the random coil. Indicating that the aggregation of the salt-induced helix is small in contrast to that of the pH-induced helix. For (Arg)_n, however, the precipitate of the helical polymers is mainly due to aggregation.     

Genetic Enhancement of Cold Tolerance by Expression of a Gene for Chloroplast [omega]-3 Fatty Acid Desaturase in Transgenic Tobacco

The increased production of trienoic fatty acids, hexadecatrienoic (16:3) and linolenic (18:3) acids, is a response connected with cold acclimation of higher plants and is thought to protect plant cells against cold damage. Transgenic tobacco (Nicotiana tabacum cv SR1) plants that contain increased levels of 16:3 and 18:3 fatty acids, and correspondingly decreased levels of their precursors, hexadecadienoic and linoleic acids, were engineered by introduction of a chloroplast [omega]-3 fatty acid desaturase gene (the fad7 gene) isolated from Arabidopsis thaliana. When exposed to 1[deg]C for 7 d and then cultured at 25[deg]C, the suppression of leaf growth observed in the wild-type plants was significantly alleviated in the transgenic plants with the fad7 gene. The low-temperature- induced chlorosis was also much reduced in the plants transformed with the fad7 gene. These results indicate that increased levels of trienoic fatty acids in genetically engineered plants enhance cold tolerance.     

Simulating Evolution by Gene Duplication

By considering the recent finding that unequal crossing over and other molecular interactions are contributing to the evolution of multigene families, a model of the origin of repetitive genes was studied by Monte Carlo simulations. Starting from a single gene copy, how genetic systems evolve was examined under unequal crossing over, random drift and natural selection. Both beneficial and deteriorating mutations were incorporated, and the latter were assumed to occur ten times more frequently than the former. Positive natural selection favors those chromosomes with more beneficial mutations in redundant copies than others in the population, but accumulation of deteriorating mutations (pseudogenes) have no effect on fitness so long as there remains a functional gene. The results imply the following: Positive natural selection is needed in order to acquire gene families with new functions. Without it, too many pseudogenes accumulate before attaining a functional gene family. There is a large fluctuation in the outcome even if parameters are the same. When unequal crossing over occurs more frequently, the system evolves more rapidly. It was also shown, under realistic values of parameters, that the genetic load for acquiring a new gene is not as large as J.B.S. Haldane suggested, but not so small as in a model in which a system for selection started from already redundant genes.     

A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication

Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the β isoform of class II PI3K (PI3K-C2β) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2β abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2β substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2β plays an indispensable role in the HCV replication cycle through the binding to HCV core protein.     

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

Regulation of gonadotropin secretion and puberty onset by neuromedin U

Neuromedin U (NMU), an anorexigenic peptide, was originally isolated from porcine spinal cord in 1985. As NMU is abundant in the anterior pituitary gland, we investigated the effects of NMU on gonadotropin secretion. Both NMU and its receptors, NMUR1 and NMUR2, were expressed in the pituitary gland. NMU suppressed LH and FSH releases from rat anterior pituitary cells. Moreover, NMU-deficient mice exhibit an early onset of vaginal opening. The LHbeta/FSHbeta ratio, which is an index of puberty onset, is high in young NMU-deficient mice. These results indicate that NMU suppresses gonadotropin secretion and regulates the onset of puberty.     

Effect of recent changes in atomic bomb survivor dosimetry on cancer mortality risk estimates

The Radiation Effects Research Foundation has recently implemented a new dosimetry system, DS02, to replace the previous system, DS86. This paper assesses the effect of the change on risk estimates for radiation-related solid cancer and leukemia mortality. The changes in dose estimates were smaller than many had anticipated, with the primary systematic change being an increase of about 10% in gamma-ray estimates for both cities. In particular, an anticipated large increase of the neutron component in Hiroshima for low-dose survivors did not materialize. However, DS02 improves on DS86 in many details, including the specifics of the radiation released by the bombs and the effects of shielding by structures and terrain. The data used here extend the last reported follow-up for solid cancers by 3 years, with a total of 10,085 deaths, and extends the follow-up for leukemia by 10 years, with a total of 296 deaths. For both solid cancer and leukemia, estimated age-time patterns and sex difference are virtually unchanged by the dosimetry revision. The estimates of solid-cancer radiation risk per sievert and the curvilinear dose response for leukemia are both decreased by about 8% by the dosimetry revision, due to the increase in the gamma-ray dose estimates. The apparent shape of the dose response is virtually unchanged by the dosimetry revision, but for solid cancers, the additional 3 years of follow-up has some effect. In particular, there is for the first time a statistically significant upward curvature for solid cancer on the restricted dose range 0-2 Sv. However, the low-dose slope of a linear-quadratic fit to that dose range should probably not be relied on for risk estimation, since that is substantially smaller than the linear slopes on ranges 0-1 Sv, 0-0.5 Sv, and 0- 0.25 Sv. Although it was anticipated that the new dosimetry system might reduce some apparent dose overestimates for Nagasaki factory workers, this did not materialize, and factory workers have significantly lower risk estimates. Whether or not one makes allowance for this, there is no statistically significant city difference in the estimated cancer risk.     

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.