Effect of light on nonphotosynthetic microorganisms. II. Photochromogenicity in genus Streptomyces.

The effect of light on the pigmentation of various strains belonging to the genus Streptomyces was investigated. It was revealed that six species of streptomycetes, S. massaporeus, S. phaeopurpureus, S. chibaensis, S. salmonicida, S. fluvissimus and S. longispororuber, were photochromogenic, i.e., these strains can be photoinduced to synthesize pigments. On the basis of these results, pigmentation in Streptomyces is discussed.     

New radioisotopic assays of argininosuccinate synthetase and argininosuccinase.

Methods were developed for the radioisotopic assay of argininosuccinate synthetase [L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5] and argininosuccinase [L-argininosuccinate arginine-lyase, EC 4.3.2.1]. The assay of argininosuccinate synthetase was based on the separation of [14C]argininosuccinate formed from aspartate and [carbamoyl-14C]citrulline in the presence of ATP from the substrate citrulline. For this, the product was converted to its anhydride form by boiling for 30 min at pH 2.0 followed by application on a column of Dowex 50W (pyridine form). Argininosuccinic anhydride was eluted with 0.3 M pyridine acetate buffer, pH 4.25, while citrulline was eluted with 0.1 M pyridine acetate buffer, pH 3.80. The assay of argininosuccinase was based on the separation of [14C]argininosuccinic acid formed from arginine and [U-14C]fumaric acid from the substrate fumarate on a column of Dowex 50W(H+ form). The argininosuccinic acid was adsorbed on the column and eluted with 1 M pyridine solution, while fumarate was not adsorbed. The distributions of these two enzymes in various organs and cell fractions were reinvestigated using these methods.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

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.     

Chemical structure of kenaf xylan

Methylation and partial acid hydrolysis of xylans from the bast and core of kenaf (Hibiscus cannabinus) showed that the main chain of these xylans consists of (1 → 4)-linked β-d-xylopyranosyl (Xylp) residues, some of which carry a -1,2-linked 4-O-methyl-glucopyranosyluronic acid (Me-GlcAp) and glucopyranosyluronic acid (GlcAp) residues as side chains. Partial hydrolysis of kenaf xylans afforded two series of aldouronic acids from aldobio- to aldotetraouronic acids. The acids of the first series composed of 4-O-Me-d-GlcAp and d-Xylp residues: 4-O-Me-GlcA-Xyl₃, 4-O-Me-GlcA-Xyl₂ and 4-O-Me-GlcA-Xyl. The second series composed of d-GlcAp and d-Xylp: GlcA-Xyl₃, GlcA-Xyl₂ and GlcA-Xyl.

In addition to these acids, another aldobiouronic acid, 4-O-(-d-GalAp)-d-Xyl was found to be present in the partial hydrolysate.

The molar ratio of GalA, GlcA, 4-O-Me-GlcA, and Xyl residues was calculated to be 1.0:2.0:9.4:119 for the bast xylan and 1.0:1.3:7.9:99.4 for the core xylan.     

Structure-activity study and analgesic efficacy of amino acid derivatives as N-type calcium channel blockers

The synthesis and structure-activity relationship (SAR) study of a novel series of N-type calcium channel blockers are described. L-Cysteine derivative 2a was found to be a potent and selective N-type calcium channel blocker with IC(50) 0.63 microM on IMR-32 assay. Compound 2a showed analgesic efficacy in the rat formalin-induced pain model by intrathecal and oral administration.     

Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus type 1-infected cell line, MT-2.

We recently found that a human T-cell leukemia virus type 1-infected cell line, MT-2, could support the replication of hepatitis C virus (HCV) (N. Kato, T. Nakazawa, T. Mizutani, and K. Shimotohno, Biochem. Biophys. Res. Commun. 206:863-869, 1995). In order to develop a culture system in which HCV replicates more efficiently, we examined the efficiency of HCV replication in cloned MT-2 cell lines by the limiting dilution method. Consequently, we obtained five clones in which intracellular positive-stranded HCV RNA could be detected until at least 21 days postinoculation (p.i.), as opposed to 15 days p.i. in uncloned MT-2 cells. MT-2C, one of the five clones which supported HCV replication up to 30 days p.i., was used for further characterization of HCV replication. Semiquantitative analysis of HCV by PCR revealed that RNA synthesis in infected cells increased after inoculation, reached a maximum level at 4 days p.i., and maintained this level until at least 11 days p.i. The 5' untranslated region of negative-stranded HCV RNA was also detected in the infected cells by two different methods with strand specificity. These results suggest that HCV replicated and multiplied in the MT-2C cells. HCV-infected MT-2C cells that were treated with antibiotics, such as G418 and hygromycin B, sustained HCV RNA for a longer period than did untreated cells. We demonstrated inhibitory effects on HCV replication by an antisense oligonucleotide complementary to the HCV core encoding region and by interferon-alpha. Furthermore, cell-free viral transmission was demonstrated by this culture system. These results suggest that our cell culture system will be useful for studying the mechanism of HCV replication, for screening antiviral agents, and for developing HCV vaccines.     

Role of the Nuclease Activities Encoded by Herpes Simplex Virus 1 UL12 in Viral Replication and Neurovirulence

Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.