Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Reduction of severe acute respiratory syndrome coronavirus-2 infectivity by admissible concentration of ozone gas and water

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Because complete elimination of SARS-CoV-2 appears difficult, decreasing the risk of transmission is important. Treatment with 0.1 and 0.05 ppm ozone gas for 10 and 20 hr, respectively, decreased SARS-CoV-2 infectivity by about 95%. The magnitude of the effect was dependent on humidity. Treatment with 1 and 2 mg/L ozone water for 10 s reduced SARS-CoV-2 infectivity by about 2 and 3 logs, respectively. Our results suggest that low-dose ozone, in the form of gas and water, is effective against SARS-CoV-2.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

cDNA microarray analysis of altered gene expression in Ara-C-treated leukemia cells

The acute lymphoblastic leukemia cell line CCRF-CEM is sensitive to Ara-C and undergoes apoptosis. In contrast, the chronic myelogenous leukemia (CML) cell line K562 is highly resistant to Ara-C, which causes the cells to differentiate into erythrocytes before undergoing apoptosis. We used cDNA microarrays to monitor the alterations in gene expression in these two cell lines under conditions leading to apoptosis or differentiation. Ara-C-treated CCRF-CEM cells were characterized by a cluster of down-regulated chaperone genes, whereas Ara-C-treated K562 cells were characterized by a cluster of up-regulated hemoglobin genes. In K562 cells, Ara-C treatment induced significant down-regulation of the asparagine synthetase gene, which is involved in resistance to L-asparaginase. Sequential treatment with Ara-C and L-asparaginase had a synergistic effect on the inhibition of K562 cell growth, and combination therapy with these two anticancer agents may prove effective in the treatment of CML, which cannot be cured by either drug alone.     

Multiform biosynthetic pathway of syringyl lignin in angiosperms

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.     

Influence of length on cytotoxicity of multi-walled carbon nanotubes against human acute monocytic leukemia cell line THP-1 in vitro and subcutaneous tissue of rats in vivo

Carbon nanotubes (CNTs) are single- or multi-cylindrical graphene structures that possess diameters of a few nanometers, while the length can be up to a few micrometers. These could have unusual toxicological properties, in that they share intermediate morphological characteristics of both fibers and nanoparticles. To date, no detailed study has been carried out to determine the effect of length on CNT cytotoxicity. In this paper, we investigated the activation of the human acute monocytic leukemia cell line THP-1 in vitro and the response in subcutaneous tissue in vivo to CNTs of different lengths. We used 220 nm and 825 nm-long CNT samples for testing, referred to as "220-CNTs" and "825-CNTs", respectively. 220-CNTs and 825-CNTs induced human monocytes in vitro, although the activity was significantly lower than that of microbial lipopeptide and lipopolysaccharide, and no activity appeared following variation in the length of CNTs. On the other hand, the degree of inflammatory response in subcutaneous tissue in rats around the 220-CNTs was slight in comparison with that around the 825-CNTs. These results indicated that the degree of inflammation around 825-CNTs was stronger than that around 220-CNTs since macrophages could envelop 220-CNTs more readily than 825-CNTs. However, no severe inflammatory response such as necrosis, degeneration or neutrophil infiltration in vivo was observed around both CNTs examined throughout the experimental period.     

Evaluation of the functional involvement of human immunodeficiency virus type 1 integrase in nuclear import of viral cDNA during acute infection

Nuclear import of viral cDNA is a critical step for establishing the proviral state of human immunodeficiency virus type 1 (HIV-1). The contribution of HIV-1 integrase (IN) to the nuclear import of viral cDNA is controversial, partly due to a lack of identification of its bona fide nuclear localization signal. In this study, to address this putative function of HIV-1 IN, the effects of mutations at key residues for viral cDNA recognition (PYNP at positions 142 to 145, K156, K159, and K160) were evaluated in the context of viral replication. During acute infection, some mutations (N144Q, PYNP>KL, and KKK>AAA) severely reduced viral gene expression to less than 1% the wild-type (WT) level. None of the mutations affected the synthesis of viral cDNA. Meanwhile, the levels of integrated viral cDNA produced by N144Q, PYNP>KL, and KKK>AAA mutants were severely reduced to less than 1% the WT level. Quantitative PCR analysis of viral cDNA in nuclei and fluorescence in situ hybridization analysis showed that these mutations significantly reduced the level of viral cDNA accumulation in nuclei. Further analysis revealed that IN proteins carrying the N144Q, PYNP>KL, and KKK>AAA mutations showed severely reduced binding to viral cDNA but kept their karyophilic properties. Taken together, these results indicate that mutations that reduced the binding of IN to viral cDNA resulted in severe impairment of virus infectivity, most likely by affecting the nuclear import of viral cDNA that proceeds integration. These results suggest that HIV-1 IN may be one of the critical constituents for the efficient nuclear import of viral cDNA.     

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.     

In vitro development of cloned embryos derived from miniature pig somatic cells after activation by ultrasound stimulation

The present study was carried out to examine the activation and development of cloned embryos produced by transferring miniature pig somatic cells into enucleated farm pig oocytes after exposing to ultrasound. The rates of the pronucleus-like structure formation and polar body-like structure extrusion in embryos exposed to ultrasound did not differ from those applied electric pulses. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from embryos activated by exposing to ultrasound was significantly (p < 0.05) higher than that obtained by applying electric pulses. The results of the present study showed that ultrasound stimulation can induce the activation and in vitro development of cloned embryos derived from miniature pig somatic cells.