Functional domains of necdin for protein-protein interaction, nuclear matrix targeting, and cell growth suppression

Necdin is a growth suppressor expressed predominantly in postmitotic neurons. The necdin gene is involved in the etiology of the genomic imprinting-associated neurodevelopmental disorder Prader-Willi syndrome and belongs to the MAGE gene family. All the MAGE family proteins contain a large homology domain termed the MAGE homology domain (MHD). We here characterize the regions of necdin required for the protein-protein interaction, nuclear matrix targeting, and cell growth suppression. The region including entire MHD (amino acids 116-280) of necdin was required for its interaction with p53, while the regions amino acids 144-184 and 191-222 within the MHD were required for both the nuclear matrix targeting and the cell growth suppression of osteosarcoma SAOS-2 cells. The amino-terminal proline-rich acidic region (amino acids 60-100) was also necessary for cell growth suppression. Tetracycline-regulatable overexpression of necdin induced growth arrest of SAOS-2 cells in a reversible manner, and the necdin-overexpressing cells showed a large, flattened morphology with double nuclei. In contrast, a necdin mutant lacking amino acids 191-222 did not induce such changes. These findings suggest that different functions of necdin are mediated via its distinct domains.     

Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan

Electrospray tandem mass spectrometry was applied to detect a series of inherited metabolic disorders during a newborn-screening pilot study and a selective screening in Japan. In our mass screening of 102,200 newborns, five patients with propionic acidemia, two with methylmalonic acidemia, two with medium-chain acyl-CoA dehydrogenase deficiency, three with citrullinemia type II, and one with phenylketonuria were identified. In a selective screening of 164 patients with symptoms mainly related to hypoglycemia and/or hyperammonemia, 12 with fatty acid oxidation disorders and six with other disorders were found. The results indicated the importance of newborn screening using this technology in Japan.     

Semi-quantitative RT-PCR analysis of photoregulated gene expression in marine diatoms

The low cell densities of diatoms and other phytoplankton in culture has precluded the use of classical RNA analysis techniques for routine studies of gene expression in large numbers of samples. This has seriously hampered studies of the basic biology of such organisms. To circumvent this problem, we have developed a high-throughput semi-quantitative RT-PCR-based protocol and used it to monitor expression of a gene encoding a fucoxanthin, chlorophyll a/c-binding protein (FCP) in the centric planktonic diatom Thalassiosira weissflogii. Analysis of FCP gene expression in dark-adapted diatom cultures revealed that mRNA levels increase 5- to 6-fold in response to white light irradiation and peak around 6 to 8 h. To determine the photoreceptors involved in this response action spectra of FCP gene expression were determined using the Okazaki large spectrograph. Responses consistent with the presence of cryptochrome-, rhodopsin- and phytochrome-type receptors could be detected. The apparent presence of phytochrome-mediated responses is of particular interest given the low fluences of red and far-red light wavelengths in the marine environment.     

Apparent diffusion time of oxygen from blood to tissue in rat cerebral cortex: implication for tissue oxygen dynamics during brain functions.

To investigate the dynamics of tissue oxygen demand and supply during brain functions, we simultaneously recorded Po(2) and local cerebral blood flow (LCBF) with an oxygen microelectrode and laser Doppler flowmetry, respectively, in rat somatosensory cortex. Electrical hindlimb stimuli were applied for 1, 2, and 5 s to vary the duration of evoked cerebral metabolic rate of oxygen (CMR(O(2))). The electrical stimulation induced a robust increase in Po(2) (4-9 Torr at peak) after an increase in LCBF (14-26% at peak). A consistent lag of approximately 1.2 s (0.6-2.3 s for individual animals) in the Po(2) relative to LCBF was found, irrespective of stimulus length. It is argued that the lag in Po(2) was predominantly caused by the time required for oxygen to diffuse through tissue. During brain functions, the supply of fresh oxygen further lagged because of the latency of LCBF onset ( approximately 0.4 s). The results indicate that the tissue oxygen supports excess demand until the arrival of fresh oxygen. However, a large drop in Po(2) was not observed, indicating that the evoked neural activity demands little extra oxygen or that the time course of excess demand is as slow as the increase in supply. Thus the dynamics of Po(2) during brain functions predominantly depend on the time course of LCBF. Possible factors influencing the lag between demand and supply are discussed, including vascular spacing, reactivity of the vessels, and diffusivity of oxygen.     

Biologically active oligodeoxyribonucleotides-IX. Synthesis and anti-HIV-1 activity of hexadeoxyribonucleotides, TGGGAG, bearing 3′- and 5′-end-modification

We have determined that hexadeoxyribonucleotides (5′TGGGAG3′), with modified aromatic groups such as a trityl group at the 5′-end, have anti-HIV-1 activity in vitro. The 6-mer bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end had the most potent activity and the least cytotoxicity. When the 3′-end of the 5′-(3,4-DBB)-modified 6-mer was substituted with a 2-hydroxyethylphosphate, a 2-hydroxyethylthiophosphate, or a methylphosphate group at the 3′-end, anti-HIV-1 activity increased. Moreover, among various 3′- and 5′-end-modified 6-mers that were tested, the 6-mer (R-95288) bearing a 3,4-DBB group at the 5′-end and a 2-hydroxyethylphosphate group at the 3′-end was the most stable, when incubated with mouse, rat, or human plasma. Therefore, R-95288 was chosen as the best candidate for possible use in therapy on the basis of its anti-HIV-1 activity.     

Lipopolysaccharide‐bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad‐spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram‐negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram‐negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α‐helical structure in a solution containing LPS. For NMR experiments, we expressed ¹⁵N‐labeled and ¹³C‐labeled CP1 in bacterial cells and successfully assigned almost all backbone and side‐chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr‐NOE) experiments in LPS. We performed ¹⁵N‐edited and ¹³C‐edited Tr‐NOE spectroscopy for CP1 bound to LPS. Tr‐NOE peaks were observed at the only C‐terminal region of CP1 in LPS. The results of structure calculation indicated that the C‐terminal region (Lys15–Gly29) formed the well‐defined α‐helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Negative Phototropism in Vaucheria terrestris Regulated by Calcium III. The Role of Calcium Characterized by Use of a High-Power Argon-Ion Laser as the Source of Unilateral Blue Light

A tip-growing Xanthophycean algal coenocyte, Vaucheria terrestrissensu Gtz, is able to change the sign of its phototropic responsefrom positive to negative as a result of its ability to sensethe fluence rate (=intensity) of unilateral blue light (BL).The mechanism that determines the sign of phototropism was investigatedusing a high-power argon-ion laser (457.9 nm) as a source ofvery strong unilateral BL. The fluence-response relationshipwas determined by changing both the fluence rate and the durationof irradiation. Positive phototropic bending was induced whenthe fluence rate of BL from the laser was below 60 W m^–2.The positive bending obeyed the reciprocity law and was notaffected by the concentrations of external Ca²⁺ ions between0.4 mM and 4.4 mM. The positive curvature decreased when thealga was exposed to a unilateral pulse of BL with a durationof 10–300 s at fluence rates higher than 60 W m^–2.The alga finally showed a deep negative curvature when eitherthe fluence rate or the duration of irradiation was furtherincreased. The inversion of the phototropic response and developmentof the negative phototropic response was greatly enhanced inthe presence of 4.4 mM Ca²⁺ ions. However, the mechanism thatdetermine the sign of phototropism seemed to require a BL pulseof longer than several seconds, even when the fluence rate wassufficiently high. The role of cytoplasmic Ca²⁺ ions in positiveand negative phototropic responses is discussed. ¹This study was carried out as part of NIBB Cooperative ResearchProgram for the Okazaki Large Spectrograph (89-513 and 90-518). ²Part of this study was reported at the XXXII Yamada Conferenceon Plant Cell Walls as Biopolymers with Physiological Functions,May 5–8, 1992, Osaka (Kataoka and Watanabe 1992).     

The Fate of Patatin and Its mRNA in Slices of Potato Tuber

The fate of patatin mRNA was investigated in slices of potatotuber since this mRNA appeared to be a potential example ofa preexisting mRNA that is involved in the rapid formation ofpolysomes which occurs in such slices. Levels of patatin, whichis the major storage protein in mature potato tubers, decreasedslightly during the first 4 h after slicing but remained constantfor the next 44 h. Analysis of products of in vitro translationshowed that patatin mRNA was present and stable in the tubercells even after several months of storage. The translationalactivity of patatin mRNA relative to total translational activityin total cellular RNA fraction increased transiently duringthe first hour and then decreased rapidly to undetectable levelswithin 6 h. By contrast, the activity in polysomal RNA fractiondecreased immediately after slicing. The difference betweenthe relative activities of patatin mRNA in total and polysomalRNA fractions during the first hour suggests that the extentof incorporation of patatin mRNA into polysomes was not in directproportion to its abundance in the cells of the slices. Additionof actinomycin D to the slices did not prevent the transientincrease in the translational activity of patatin mRNA in totalRNA fraction at 1 h, indicating that the transient increasewas not due to synthesis of patatin mRNA de novo after slicing.However, the inhibitor prevented the degradation of patatinmRNA in the slices. This result indicates that the synthesisof new mRNAs is necessary for the degradation of patatin mRNA. ¹Present address: Aburahi Laboratories, Shionogi and Co., Ltd.,Koka-cho, Shiga, 520-34 Japan (Received June 30, 1989; Accepted May 9, 1990)     

Microspectrofluorometry of the autofluorescent flagellum in phototactic brown algal zoids

Summary Posterior flagella of zoids ofScytosiphon lomemaria andChorda filum (Phaeophyceae, Chromophyta) were isolated and subjected to microspectrofluorometry using a high sensitivity microspectrofluorometer in order to characterize the flagellar autofluorescent substances. Vigorous agitation in a Hypertonic medium yielded preparations of largely intact flagella retaining detectable green flagellar autofluorescence. Under 380–425 nm excitation light, maximum emission of flagellar autofluorescence was observed at 495 nm, whereas under 400–440 nm excitation light fluorescence shifted to around 510 nm. Comparison of these spectra with the fluorescence spectra of 4,5-cyclic FMN isolated from fertile thalli ofS. lomentaria, and of 6-carboxypterin suggested that two (or more) different fluorescent substances (presumably a flavin and a pterin) are present in the flagella.Abbreviations DTT dithiothreitol - FMN flavin mononucleotide - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) - PEG polyethylene glycol - PFB paraflagellar body - Tris tris(hydroxymethyl) aminomethane. Dedicated to Professors Masakazu Tatewaki and Tadao Yoshida on the occasion of their academic retirement