Versatile Method for Evaluating a Cell Culture by Various Morphological Techniques

Cellulose acetate is a versatile material for evaluating cells grown under identical conditions by various morphological techniques. This inexpensive material is transparent, easily cut to size and shape, nontoxic to cell cultures, and resistant to most chemicals used in histochemistry and in scanning and transmission electron microscopy. Samples may be obtained during and after the culture process. Cellulose acetate slides can be mounted directly over glass slides for direct observation and are easily peeled off plastic blocks for electron microscopy, leaving the cells behind. Relative disadvantages include its autofluorescence and a tendency to soften in strong acids or pure solutions of organic solvents such as xylene and propylene oxide.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Solaniol, a Toxic Metabolite of Fusarium solani

Fusarium solani M-1-1 isolated from moldy bean hulls produces T-2 toxin, diacetoxyscirpenol, and a new toxic trichothecene, solaniol, in Czapek-Dox-peptone medium.     

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Plant reproductive intervals and pollinators in the aseasonal tropics: a new model

What factors determine reproductive intervals and modes of pollination in plants of the aseasonal tropics? To answer this general question, we present a new explanation for some community patterns of plant reproductive intervals and pollinators observed in a lowland dipterocarp forest in Sarawak, Malaysia, using a mathematical model featuring different display effects for different types of pollinators. Predictions from the model matched with the following observed patterns: (i) flowering intervals were different among forest strata (forest floor < understorey < canopy < subcanopy and emergent), and not in the exact order of stratum height; (ii) among generalist pollinators, the proportion of social foragers was maximum in intermediate forest stratum; and (iii) plants pollinated by specialist pollinators were found on the forest floor and in gaps.     

(4-Piperidinyl)-piperazine: A new platform for acetyl-CoA carboxylase inhibitors

Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the synthesis and structure–activity relationships of a series of disubstituted (4-piperidinyl)-piperazine derivatives as a new platform for ACC1/2 non-selective inhibitors.     

Photosynthesis–nitrogen relationships in species at different altitudes on Mount Kinabalu, Malaysia

In situ photosynthetic nitrogen-use efficiency (PNUE, photosynthetic capacity per unit leaf nitrogen) was investigated in species that commonly distributed at different altitudes (600–3700m above sea level) on Mount Kinabalu. Photosynthetic nitrogen-use efficiency was lower in species at higher altitudes, with a mean PNUE at 3700m being one-third as large as that at 600m. This difference in PNUE was larger than that explained by the biochemical response to lower air pressures only. Across altitudes a negative correlation between ¹³C abundance (¹³C) and PNUE was found. Species at higher altitudes tended to have higher ¹³C, suggesting that they had a lower conductance for CO₂ diffusion from the air to chloroplasts. The lower conductance might be responsible for the lower PNUE in species at higher altitudes. Although leaf nitrogen content per unit area tended to be higher at higher altitudes, it did not seem to contribute to increasing photosynthetic rates. Thus, the idea that a higher nitrogen content at higher altitudes is a compensation for a lower PNUE was not supported. In contrast to the large difference in PNUE among altitudes, PNUE tended to converge within a narrow range among species growing at the same altitude.     

Discovery and analysis of cofactor-dependent phosphoglycerate mutase homologs as novel phosphoserine phosphatases in Hydrogenobacter thermophilus

Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. PSPs, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. However, certain organisms, particularly bacteria, lack a classical PSP gene, although they appear to possess a functional phosphoserine synthetic pathway. The apparent lack of a PSP ortholog in Hydrogenobacter thermophilus, an obligately chemolithoautotrophic and thermophilic bacterium, represented a missing link in serine anabolism because our previous study suggested that serine should be synthesized from phosphoserine. Here, we detected PSP activity in cell-free extracts of H. thermophilus and purified two proteins with PSP activity. Surprisingly, these proteins belonged to the histidine phosphatase superfamily and had been annotated as cofactor-dependent phosphoglycerate mutase (dPGM). However, because they possessed neither mutase activity nor the residues important for the activity, we defined these proteins as novel-type PSPs. Considering the strict substrate specificity toward l-phosphoserine, kinetic parameters, and PSP activity levels in cell-free extracts, these proteins were strongly suggested to function as PSPs in vivo. We also detected PSP activity from "dPGM-like" proteins of Thermus thermophilus and Arabidopsis thaliana, suggesting that PSP activity catalyzed by dPGM-like proteins may be distributed among a broad range of organisms. In fact, a number of bacterial genera, including Firmicutes and Cyanobacteria, were proposed to be strong candidates for possessing this novel type of PSP. These findings will help to identify the missing link in serine anabolism.     

Molecular epidemiology of adenovirus type 8 (Ad 8) in Taiwan: four subtypes recovered during the period of 1980-1981 from patients with epidemic keratoconjunctivitis

Adenovirus type 8 (Ad 8) has been the major and important causative agent of epidemic keratoconjunctivitis (EKC). By enzymatic cleavage analysis with five endonucleases, PstI, HindIII, BamHI, SalI and SstI, 27 out of 149 Ad 8 isolates recovered from patients with EKC during the period from July, 1980 to July, 1981 in Kaohsiung, Taiwan, were studied. By cleavage patterns, 27 Ad 8 isolates in Kaohsiung were classified into four subtypes which were found to be different from the subtypes (Ad 8A, Ad 8B) prevalent in Sapporo (1).     

Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells

In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, α-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [³⁵S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, β subunit (Cct2); heterogenous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5′-UTR of Cct2 mRNA.