Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings XI. Inhibition of RNA Synthesis by 4-Thiouridine Nucleotides during Germination of Radish Seeds

The effects of 4-thiouridine and its metabolites on RNA synthesisin radish (Raphanus sativus) cotyledons were investigated. The4-thiouridine nucleotides, 4-thioUTP and 4-thioUDP, were foundto inhibit DNA-dependent RNA polymerase activities of isolatednuclei and of chloroplasts from radish cotyledons. These inhibitorsappeared to compete with UTP for its binding to RNA polymerase.Neither 4-thiouridine nor 4-thioUMP inhibited RNA polymeraseactivities. Reduced RNA accumulation accompanied by the inhibition of protochlorophyllideaccumulation was observed in cotyledons of dark-grown radishseedlings germinated and grown with 4-thiouridine. On the otherhand, 4-thiouridine had no effect on chloroplast developmentin the control seedlings germinated and grown without 4-thiouridine.These results suggest that the inhibition of chloroplast developmentby 4-thiouridine may in part be due to the inhibition of RNAsynthesis by 4-thiouridine nucleotide during germination, resultingin inhibition of etioplast development. (Received December 9, 1985; Accepted June 26, 1986)     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and beta-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H2O2 system. But they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Penicillin-binding proteins in Mycobacterium smegmatis

Abstract The penicillin-binding proteins (PBPs) of Mycobacterium smegmatis were studied. Five PBPs ranging in M _r from approx. 114000 to 25000 were detected in the cytoplasmic membrane. The affinities of the PBPs for selected β-lactam antibiotics were determined. Most of the antibiotics bound to PBPs 3 and 4 at low concentrations. A penicillin-susceptible mutant and a cefmetazole-resistant mutant were isolated by selection in vitro. The PBPs of these mutants were identical to those of the parent strain. The affinity of cefmetazole for the individual PBPs was identical in each mutant.     

Structural Differences between the Avian and Human H7N9 Hemagglutinin Proteins Are Attributable to Modifications in Salt Bridge Formation: A Computational Study with Implications in Viral Evolution

Influenza A hemagglutinin (HA) is a homotrimeric glycoprotein composed of a fibrous globular stem supporting a globular head containing three sialic acid binding sites responsible for infection. The H7N9 strain has consistently infected an avian host, however, the novel 2013 strain is now capable of infecting a human host which would imply that the HA in both strains structurally differ. A better understanding of the structural differences between the avian and human H7N9 strains may shed light into viral evolution and transmissibility. In this study, we elucidated the structural differences between the avian and human H7N9 strains. Throughout the study, we generated HA homology models, verified the quality of each model, superimposed HA homology models to determine structural differences, and, likewise, elucidated the probable cause for these structural differences. We detected two different types of structural differences between the novel H7N9 human and representative avian strains, wherein, one type (Pattern-1) showed three non-overlapping regions while the other type (Pattern-2) showed only one non-overlapping region. In addition, we found that superimposed HA homology models exhibiting Pattern-1 contain three non-overlapping regions designated as: Region-1 (S157₁-A160₁); Region-3 (R262₁-S265₁); and Region-4 (S270₁-D281₁), whereas, superimposed HA homology models showing Pattern-2 only contain one non-overlapping region designated as Region-2 (S137₁-S145₁). We attributed the two patterns we observed to either the presence of salt bridges involving the E114₁ residue or absence of the R141₁:D77₁ salt bridge. Interestingly, comparison between the human H7N7 and H7N9 HA homology models showed high structural similarity. We propose that the putative absence of the R141₁:D77₁ salt bridge coupled with the putative presence of the E114₁:R262₁ and E114₁:K264₁ salt bridges found in the 2013 H7N9 HA homology model is associated to human-type receptor binding. This highlights the possible significance of HA salt bridge formation modifications in viral infectivity, immune escape, transmissibility and evolution.     

Biochemical genetic evidence for a hybrid origin of spined loach, Cobitis taenia taenia, in Japan

Starch gel electrophoresis and isoelectric focusing were performed on sarcoplasmic proteins from three taxa of cobitid fish in Japan, Cobitis biwae , C. taenia striata and C. l. taenia . These taxa are hardly distinguishable from each other by external appearance or morphological characters. Electrophoretic patterns of sarcoplasmic proteins from white muscle suggested fixed allelic differences between C. biwae and C. t. striata at two protein-coding loci (parvalbumin and creatine kinase). The third taxon, C. t. taenia , showed composite banding patterns with respect to the above two proteins, suggesting C. t, taenia originated from hybridization between C. biwae and C, t. striata . Previously, C. t, taenia and C. t. striata were believed to have shared a common ancestor subsequent to their divergence from C. biwae .     

Development of LC-MS Method for Detection of Mutant Uromodulin Protein

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 → 381, 471, 567, and 679 for GWHWE and m/z 688 → 381, 445, 541, and 653 for GWHC*E) were detected.     

MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients

MicroRNAs (miRNAs) are very short (18–24 nucleotides) nucleic acids that are expressed in a number of biological tissues and have been shown to be more resistant to extreme temperatures and pH compared to longer RNA molecules, like mRNAs. As miRNAs contribute to diverse biological process and respond to various kinds of cellular stress, their utility as diagnostic biomarkers and/or therapeutic targets has recently been explored. Here, we have evaluated the usefulness of miRNA quantification during postmortem examination of cardiac tissue from acute myocardial infarction (AMI) patients. Cardiac tissue was collected within one week of the patient’s death and either frozen (19 samples) or fixed in formalin for up to three years (36 samples). RNA integrity was evaluated with an electropherogram, and it appears that longer RNAs are fragmented after death in the long-term fixed samples. Quantitative PCR was also performed for seven miRNAs and three other small RNAs in order to determine the appropriate controls for our postmortem analysis. Our data indicate that miR-191 and miR-26b are more suitable than the other types of small RNA molecules as they are stably detected after death and long-term fixation. Further, we also applied our quantitation method, using these endogenous controls, to evaluate the expression of three previously identified miRNA biomarkers, miR-1, miR-208b, and miR-499a, in formalin-fixed tissues from AMI patients. Although miR-1 and miR-208b decreased (1.4-fold) and increased (1.2-fold), respectively, in the AMI samples compared to the controls, the significance of these changes was limited by our sample size. In contrast, the relative level of miR-499a was significantly decreased in the AMI samples (2.1-fold). This study highlights the stability of miRNAs after death and long-term fixation, validating their use as reliable biomarkers for AMI during postmortem examination.     

Studies on Herbicides and Plant Growth Regulators

With the aim of obtaining plant growth regulators, we have synthesized 1-aryloxyacylbenzimidazoles, 1-aryloxyacylindazoles, 2-aryloxyacyl-4,5,6,7-tetrahydroindazoles and 1-aryloxyacylbenzotriazoles by the acylation of azoles, and 1-aryloxyacyl-4,5,6,7-tetrahydroindazoles by the reaction of aryloxyacylhydrazines with 2-(hydroxymethylene)cyclohexanone. The structures of these compounds are discussed on the basis of nuclear magnetic resonance data.