Position of the amino terminus of myosin light chain 1 and light chain 2 determined by electron microscopy with monoclonal antibody

The position of the N terminus of myosin light chain 1 (LC1) and myosin light chain 2 (LC2) of rabbit skeletal muscle was mapped on the myosin head with a monoclonal antibody (SI304), which recognized the amino acid sequence N-trimethylalanyl-prolyl-lysyl-lysyl at the N terminus of LC1 and LC2. The complex of the antibody and myosin was observed by electron microscopy. By selective cleavage of the N terminus of LC1 or LC2 with papain or chymotrypsin, the position of the N terminus of LC1 and LC2 was determined separately. The N terminus of LC2 is located at the head-rod junction. The N terminus of LC1 is 11 nm (+/- 3 nm, standard deviation) from the head-rod junction. This position is near the actin-binding site of the myosin head.     

Antitumor, antiviral and natural killer-inducing activities of human recombinant interferon-alpha in the guinea pig

It was found that human recombinant interferon-alpha (Hu rIFN-alpha) was biologically active in the guinea pig. Namely, intratumor injection of either Hu rIFN-alpha A or A/D regressed tumors and prevented metastasis of guinea-pig line 10 tumor. These rIFNs augmented splenic natural killer activity of the guinea pig and reduced the cytopathic effect of virus on guinea-pig cells. Hu IFN-gamma showed no such activity.     

Structure Analysis of Proteins and Peptides by Difference Circular Dichroism Spectroscopy

The Protein Journal - Difference circular dichroism (CD) spectroscopy was used here to characterize changes in structure of flexible peptides upon altering their environments. Environmental changes...     

Ultrasound Enhances Retrovirus‐Mediated Gene Transfer

Abstract

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus‐mediated gene transfer efficiency.

Retrovirus‐mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with β‐galactosidase (β‐Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm² to 4.0 watts/cm²) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated.

Below 1.0 watts/cm² and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm² of an ultrasound resulted in significant increases in retrovirus‐mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6‐fold, 4.8‐fold, 2.3‐fold, and 3.2‐fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, β‐Gal activities were also increased by the retrovirus with ultrasound exposure in these cells.

Adjunctive ultrasound exposure was associated with enhanced retrovirus‐mediated transgene expression in vitro. Ultrasound associated local gene therapy has potential for not only plasmid‐DNA‐, but also retrovirus‐mediated gene transfer.     

Heterotrimeric G protein β subunit GPB1 and MAP kinase MPK1 regulate hyphal growth and female fertility in Fusarium sacchari

Heterotrimeric GTP-binding proteins (G proteins) and mitogen-activated protein kinase (MAPK) cascades involve vegetative hyphal growth, development of infection-related structure, colonization in host plant and female fertility in phytopathogenic ascomycete fungi. In this study, a heterotrimeric G protein β subunit (Gβ), GPB1, and MAPK, MPK1, were characterized from Fusarium sacchari (= Gibberella sacchari; mating population B of the G. fujikuroi-species complex). GPB1 and MPK1 showed high homology to known Gβ and Fus3/Kss1 MAP kinases of other filamentous ascomycetes, respectively. Disruption (Δ) of gpb1 suppressed hyphal branching and accelerated aerial hyphae formation in F. sacchari. Oppositely, disruption of mpk1 caused delayed aerial hyphae formation. These indicated that GPB1 regulates vegetative hyphal growth negatively, and MPK1 does positively in F. sacchari. Both Δgpb1 and Δmpk1 showed female sterility. Level of intracellular cAMP in Δgpb1 was lower than wild type. Exogenous cyclic AMP (cAMP) partially restored enhanced aerial hyphae formation. These suggested that abnormal hyphal growth was caused by depletion of intracellular cAMP in Δgpb1. cAMP has been reported to suppress development of perithecia in crossing between wild type strains. Thus, precise regulation of intracellular cAMP level via Gβ/MAPK is essential for normal hyphal growth and fertility.     

GPAT2, a mitochondrial outer membrane protein,in piRNA biogenesis in germline stem cells

piRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.     

Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit–subunit interactions through mutations.     

Parasporin 1Ac2, a Novel Cytotoxic Crystal Protein Isolated from Bacillus thuringiensis B0462 Strain

Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S¹¹³–R²⁵⁰) and 60 kDa (I²⁵¹–S⁷⁷⁷) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.