Elongation and shortening of time required for entry into S phase after release from G 1 and G 0 arrests in temperature-sensitive mutants of rat 3Y1 Cells

Temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts representing four separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly in the G1 phase when cells of randomly proliferating population at 33.8 degrees C are shifted to 39.8 degrees C (temperature arrest). We examined the time lag of the cellular entry into the S phase after release at 33.8 degrees C, both from the temperature arrest and from the arrest at 33.8 degrees C at a confluent cell density (density arrest). In the temperature-arrested cells, as the duration of temperature arrest increased, the time lag of entry into S phase after shift down to 33.8 degrees C was prolonged, in all four mutants. These observations suggest that the four different functional lesions, each causing arrest in the G1 phase, are also responsible for prolongation of the time lag of entry into the S phase in cells arrested in the G1 phase. The prolongation of the time lag in the temperature-arrested cultures was accelerated at a higher cell density, in medium supplemented with a lower concentration of serum, and at a higher restrictive temperature. In the density-arrested cells, as the duration of pre-exposure to 39.8 degrees C was increased, the time lag of entry into S phase at 33.8 degrees C after release from the arrest was drastically prolonged, in all four mutants. In 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203, when the density-arrested cells were prestimulated by serum at 39.8 degrees C for various periods of time, the time lag of entry into S phase after release from the density arrest at 33.8 degrees C was initially shortened, and then, prolonged progressively as the period of prestimulation increased. These findings, taken together with other data, show that all four ts defects affect cells in states ranging from the deeper resting to mid- or late-G1 phase. It is suggested that events represented by these four mutants are required for entry into the S phase and normally operate in parallel but not in sequence in cells in states ranging from the deeper resting to the mid- or late-G1 phases, though they may affect each other.     

Syntheses of ( + )-Phrymarolin II and Its Stereoisomers

Phrymarolin II, a unique naturally-occurring lignan having a 1,2-dioxygenated 3,7-dioxabicyclo[3.3.0]octane skeleton, was synthesized by the reaction of sesamol, in the presence of cadmium carbonate, with 1-acetoxy-2-chloro-6-(2′-methoxy-4′,5′-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane. The latter was prepared through 13 steps starting from an aldol condensation of β-vinyl-γ-butyrolactone with 2-methoxy-4,5-methylenedioxybenzaldehyde. Three diastereomers of phrymarolin II were also obtained.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.     

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.     

Glucagon-like peptide-1 (GLP-1) inhibits advanced glycation end product (AGE)-induced up-regulation of VCAM-1 mRNA levels in endothelial cells by suppressing AGE receptor (RAGE) expression

Glucagon-like peptide-1 (GLP-1) is one of the incretins, a gut hormone secreted from L cells in the intestine in response to food intake. It has been proposed as a potential therapeutic target for the treatment of patients with type 2 diabetes. However, the direct effects of GLP-1 on vascular injury in diabetes are largely unknown. Since there is a growing body of evidence that advanced glycation end products (AGE) and their receptor RAGE axis plays an important role in vascular complications in diabetes, this study investigated whether and how GLP-1 blocked the deleterious effects of AGE on human umbilical vein endothelial cells (HUVEC). GLP-1 receptor (GLP-1R) was expressed in HUVEC. GLP-1 dose-dependently inhibited RAGE gene expression in HUVEC, which was blocked by small interfering RNAs raised against GLP-1R. An analogue of cyclic AMP also decreased RAGE mRNA level in HUVEC. Further, GLP-1 decreased reactive oxygen species generation and subsequently reduced vascular cell adhesion molecule-1 mRNA levels in AGE-exposed HUVEC. Our present study suggests that GLP-1 directly acts on HUVEC via GLP-1R and it could work as an anti-inflammatory agent against AGE by reducing RAGE expression via activation of cyclic AMP pathways.     

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.     

Fat Synthesis in Unicellular Algae

The cultural conditions of Chlorella ellipsoidea were studied with special reference to lipide accumulation. It was ascertained that there is a close relation between the lipide content and the nitrogen content of cells. Cultivation with the nitrogen-free medium was applied to produce nitrogen deficient cells, and in this culture were studied the effects of light intensity, algal density and depth of algel suspension upon lipide accumulation. The annual yield of fat by mass-culture was roughly estimated.     

Speech Misperception: Speaking and Seeing Interfere Differently with Hearing

Speech perception is thought to be linked to speech motor production. This linkage is considered to mediate multimodal aspects of speech perception, such as audio-visual and audio-tactile integration. However, direct coupling between articulatory movement and auditory perception has been little studied. The present study reveals a clear dissociation between the effects of a listener’s own speech action and the effects of viewing another’s speech movements on the perception of auditory phonemes. We assessed the intelligibility of the syllables [pa], [ta], and [ka] when listeners silently and simultaneously articulated syllables that were congruent/incongruent with the syllables they heard. The intelligibility was compared with a condition where the listeners simultaneously watched another’s mouth producing congruent/incongruent syllables, but did not articulate. The intelligibility of [ta] and [ka] were degraded by articulating [ka] and [ta] respectively, which are associated with the same primary articulator (tongue) as the heard syllables. But they were not affected by articulating [pa], which is associated with a different primary articulator (lips) from the heard syllables. In contrast, the intelligibility of [ta] and [ka] was degraded by watching the production of [pa]. These results indicate that the articulatory-induced distortion of speech perception occurs in an articulator-specific manner while visually induced distortion does not. The articulator-specific nature of the auditory-motor interaction in speech perception suggests that speech motor processing directly contributes to our ability to hear speech.