Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.     

Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli.

Ohyama et al. previously isolated Escherichia coli mutant RS1, which had a negligible activity for sodium ion extrusion at alkaline pH (T. Ohyama, R. Imaizumi, K. Igarashi, and H. Kobayashi, J. Bacteriol. 174:7743-7749, 1992). Our present study showed that the mutation of RS1 was compensated for by a cloned chaA gene. It has been proposed that sodium ion extrusion by ChaA is prevented under physiological conditions (D. M. Ivey, A. A. Guffanti, J. Zemsky, E. Pinner, R. Karpel, E. Padan, S. Schuldiner, and T. A. Krulwich, J. Biol. Chem. 268:11296-11303, 1993). In order to clarify the physiological role of chaA in sodium ion circulation at alkaline pH, we constructed a delta chaA mutant. The resultant mutant, TO112, deficient in both nhaA and chaA, was unable to grow at pH 8.5 in medium containing 0.1 M sodium chloride and had negligible sodium ion extrusion activity. However, TO112 grew at pH 7.0 in medium containing 0.4 M sodium chloride. Sodium ions were extruded from TO112 cells at neutral pH. The extrusion activity at pH 7.5 was greatly reduced by the deletion of nhaB. These data demonstrate that the activity of nhaB is low at high pH and that ChaA extrudes sodium ions at alkaline pH. The uptake of calcium ions by everted membrane vesicles prepared from the delta chaA mutant TO110 was 60% of the activity observed in the vesicles of the wild-type strain at pH 8.5, but the activity at neutral pH was not reduced by the deletion of chaA. Therefore, it was also suggested that ChaA plays a role in calcium ion circulation at alkaline pH.     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.     

Group I introns in the liverwort mitochondrial genome: the gene coding for subunit 1 of cytochrome oxidase shares five intron positions with its fungal counterparts.

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, contains thirty-two introns. Twenty-five of these introns possess the characteristic secondary structures and consensus sequences of group II introns. The remaining seven are group I introns, six of which happen to interrupt the gene coding for subunit 1 of cytochrome oxidase (cox1). Interestingly, the insertion sites of one group II and four group I introns in the cox1 gene coincide with those of the respective fungal mitochondrial interns. Moreover, comparison of the four group I introns with their fungal counterparts shows that group I introns inserted at identical genomic sites in different organisms are indeed related to one another, in terms of the peptide sequences generated from the complete or fragmental ORFs encoded by these introns. At the same time, the liverwort introns turned out to be more divergent from their fungal cognates than the latter are from one another. We therefore conclude that vertical transmission from a common ancestor organism is the simplest explanation for the presence of cognate introns in liverwort and fungal mitochondrial genomes.     

A novel cDNA clone for acid invertase in tomato fruit.

The sequence of a novel cDNA clone, Aiv-1, for tomato acid invertase was similar to that of TIV1 (Klann et al., 1992) for the enzyme except for a unique intron-like insertion. It is considered that Aiv-1 is derived from either an alternatively spliced mRNA for an isozyme or a pre-mRNA of TIV1.     

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.     

Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non‐transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.     

Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)₁₅ and b(CA)₁₅]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F₂ population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.