Formation of spray-dried powder of S-adenosyl-L-methionine

S-Adenosyl-L -methionine (SAM) is an essential metabolite in all living organisms. In clinical research, SAM has also been suggested as a chemotherapeutic agent in various diseases. The main problem of SAM is its instability at high temperatures, at neutral and alkaline pH, and in the presence of humidity. SAM retention in spray-dried powder was determined under various conditions of spray-drying. The highest SAM retention was obtained when maltodextrin (dextrose equivalent, DE, of 25) was used as the carrier solid with the SAM feed liquid at pH 4.0. The water content in the powder had a significant effect on the stability of SAM. SAM powder with lower water content exhibited higher stability.     

Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin.

The synthesis of a 5'-O-BzH-2'-O-ACE-protected-3-methyluridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine. Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S ribosomal RNA were generated. Modifications were present at positions 1911, 1915, and 1917. The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy     

Associations between White Matter Hyperintensities and β Amyloid on Integrity of Projection,Association, and Limbic Fiber Tracts Measured with Diffusion Tensor MRI

The goal of this study was to assess the relationship between Aβ deposition and white matter pathology (i.e., white matter hyperintensities, WMH) on microstructural integrity of the white matter. Fifty-seven participants (mean age: 78±7 years) from an ongoing multi-site research program who spanned the spectrum of normal to mild cognitive impairment (Clinical dementia rating 0–0.5) and low to high risk factors for arteriosclerosis and WMH pathology (defined as WMH volume >0.5% total intracranial volume) were assessed with positron emission tomography (PET) with Pittsburg compound B (PiB) and magnetic resonance and diffusion tensor imaging (DTI). Multivariate analysis of covariance were used to investigate the relationship between Aβ deposition and WMH pathology on fractional anisotropy (FA) from 9 tracts of interest (i.e., corona radiata, internal capsule, cingulum, parahippocampal white matter, corpus callosum, superior longitudinal, superior and inferior front-occipital fasciculi, and fornix). WMH pathology was associated with reduced FA in projection (i.e., internal capsule and corona radiate) and association (i.e., superior longitudinal, superior and inferior fronto-occipital fasciculi) fiber tracts. Aβ deposition (i.e., PiB positivity) was associated with reduced FA in the fornix and splenium of the corpus callosum. There were interactions between PiB and WMH pathology in the internal capsule and parahippocampal white matter, where Aβ deposition reduced FA more among subjects with WMH pathology than those without. However, accounting for apoE ε4 genotype rendered these interactions insignificant. Although this finding suggests that apoE4 may increase amyloid deposition, both in the parenchyma (resulting in PiB positivity) and in blood vessels (resulting in amyloid angiopathy and WMH pathology), and that these two factors together may be associated with compromised white matter microstructural integrity in multiple brain regions, additional studies with a longitudinal design will be necessary to resolve this issue.     

Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).     

DNA polymerase-primase from embryos of Drosophila melanogaster. The DNA polymerase subunit

The DNA polymerase-primase from Drosophila melanogaster has been separated into its constituent polymerase and primase subunits by sedimentation in glycerol gradients containing 50% ethylene glycol. Both activities have been obtained in good yield. The properties of the 182-kDa polymerase subunit are similar to those of the intact four-subunit enzyme. However, there are three significant differences. (i) The polymerase activity of the 182-kDa subunit shows an increased thermolability; (ii) the pause sites during replication of singly primed, single-stranded circular DNA by the 182-kDa subunit are altered; and (iii) unlike the intact enzyme, the 182-kDa subunit is highly processive in the presence of the single-stranded DNA-binding protein of Escherichia coli.     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.     

Transport of proteins into yeast mitochondria

The amino-terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix-targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop-transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron-sulphur protein of the cytochrome bc1 complex). Matrix-targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix-targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix-targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein. Transport of proteins across mitochrondrial membranes requires a membrane potential, ATP, and a 45-kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP-dependent unfolding of the precursor protein.     

噬菌体T7基因6.5和6.7的克隆及其缺失变种的构建

 用适当的限制性内切酶,将噬菌体T7基因6.5和6.7从整个噬菌体T7基因组中分离出来,插入到质粒pBR322中去,转化E.Coli HMS174,筛选出这两个基因的成功克隆。运用同样手段,从整个噬菌体T7基因组中分离出含有部分基因6编码序列,而不含基因6.5和6.7编码序列的T7DNA片段,插入到pBR322的衍生质粒中去,转化Ecoli C1757,再用含有基因6和基因7的双突变噬菌体T7去感染这一转化菌,通过同源交叉而得到缺失基因6.5和6.7的噬菌体T7缺失变种。这种噬菌体只能在载有噬菌体T7基因6.5和6.7,或者只载有基因6.7质粒的寄主中增殖。通过噬菌体结构蛋白电泳分析证明,这种噬菌体丢失了野生型菌体T7所具有的两条结构蛋白带。     

Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.