Involvement of polyamines in gibberellin-induced development of seedless grape berries

The roles of polyamines (PAs) in the development of seedless grape berries induced by gibberellin (GA₃) was investigated. The development of seedless grape berries was stimulated by the application of putrescine (Put), but not by that of spermidine (Spd) and spermine (Spm), regardless of the presence of GA₃. At harvest, the fresh weight of seedless grape berries treated with 500 ppm Put + 25 ppm GA₃ and 500 ppm Put increased to 111 and 112%, respectively, of the control. Treatment with methylglyoxal-bis (guanyl hydrazone), a potent inhibitor of S-adenosylmethionine decarboxylase that plays a role in Spd and Spm synthesis, did not affect the development of seedless grape berries induced by 100 ppm GA₃. The application of 100 ppm GA₃ significantly increased endogenous free Put levels. Levels of free Spd and Spm were not affected by GA₃. Although the levels of endogenous perchloric acid insoluble bound PAs were higher than those of free PAs, obvious changes in the levels of bound PAs were not observed. These results indicate that free Put is implicated in the development of seedless grape berries induced by GA₃.     

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.     

The antimalarial drugs chloroquine and primaquine inhibit pyridoxal kinase,an essential enzyme for vitamin B6 production

Quinoline derivatives such as chloroquine and primaquine are widely used for the treatment of malaria. These drugs are also used for the treatment of trypanosomiasis, and more recently for cancer therapy. However, molecular target(s) of these drugs remain unclear. In this study, we have identified human pyridoxal kinase as a binding protein of primaquine. Primaquine inhibited pyridoxal kinases of malaria, trypanosome and human, while chloroquine inhibited only malaria pyridoxal kinase. Thus, we have identified pyridoxal kinase as a possible target molecule of the antimalarial drugs chloroquine and primaquine.     

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Δ52 in an αC–βC loop. Double deletion mutants showed that the loop αC–βC has nonadditive couplings with the βF–βG and βG–βH loops regarding stability. Single deletion to the loops αC–βC or βC–βD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.     

Cyclin A2 confers cisplatin resistance to endometrial carcinoma cells via up‐regulation of an Akt‐binding protein,periplakin

Although overexpression of cyclin A2 is reportedly an indicator of a poor prognosis of various malignancies including endometrial carcinoma, its molecular mechanism remains undetermined. To address this issue, we examined the effect of cyclin A2 on the development of resistance to chemotherapeutic drugs. The expression of cyclin A2 protein was increased in advanced‐stage and chemotherapy‐refractory stage endometrial carcinomas compared with that in early‐stage tumours. The expression levels of cyclin A2 in endometrial carcinoma cell lines correlated positively with the IC₅₀ for cisplatin. Endometrial carcinoma HHUA cells that overexpressed cyclin A2 showed increased resistance to cisplatin in vitro and in vivo, via the activation of a survival pathway, the inositol‐3 phosphate kinase (PI3K) cascade. The use of a cDNA microarray identified an Akt‐binding protein, periplakin, as a novel target of cyclin A2. The cyclin A2‐induced up‐regulation of periplakin was mediated via direct binding of Sp1 to the promoter that was activated by cyclin A2 along with chromatin remodelling involving CBP/p300, and the siRNA‐mediated silencing of periplakin suppressed the PI3K pathway. These results indicate cyclin A2 to be involved in the acquisition of aggressive behaviour of tumour cells through the activation of PI3K by cyclin A2‐induced periplakin, and to be a promising therapeutic target.     

Bowman-birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. japonica cells.     

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.     

Effects of disinhibition of neurons in the dorsomedial hypothalamus on central respiratory drive

Neurons within the dorsomedial hypothalamus (DMH) play a critical role in subserving the cardiovascular and neuroendocrine response to psychological stress. An increase in respiratory activity is also a characteristic feature of the physiological response to psychological stress, but there have been few studies of the role of DMH neurons in regulating respiratory activity. In this study we determined the effects of activation of DMH neurons on respiratory activity (assessed by measuring phrenic nerve activity, PNA) and the relationship between evoked changes in respiratory activity and changes in sympathetic vasomotor activity in spontaneously breathing urethane-anesthetized rats. Microinjections of bicuculline (4-40 pmol in 20 nl) into the DMH evoked dose-dependent increases in PNA burst frequency and amplitude. These were accompanied by dose-dependent decreases in mean tracheal CO(2) levels, indicative of hyperventilation. In control experiments, microinjections of bicuculline into sites adjacent to the DMH evoked much smaller or no changes in PNA. In experiments where renal sympathetic nerve activity (RSNA) was also measured, cycle-triggered averaging revealed that RSNA under resting conditions was partly correlated with the PNA, but in response to DMH disinhibition there was no consistent change in the amplitude of the respiratory-related variations in RSNA. The results indicate that DMH neurons can exert a powerful stimulatory effect on respiratory activity, causing hyperventilation. This is not associated with an increase in the degree of coupling between PNA and RSNA, indicating that the DMH-evoked increase in RSNA is not a consequence of increased central respiratory drive.     

Asymmetric coiled-coil structure with Guanine nucleotide exchange activity

Vesicular traffic during exocytosis is regulated by Rab GTPase, Sec4p in yeast, which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. The GEF activity is localized in the N-terminal 160 residues of Sec2p, which lacks sequence similarity with any other GEFs with known structures, and thereby the guanine nucleotide exchange mechanism by Sec2p remains unknown. Here, we report the crystal structure of the Sec2p GEF domain at 3.0 A resolution. The structure unexpectedly consists of a homodimeric, parallel coiled coil that extends over 180 A. Pull-down and guanine nucleotide exchange analyses on a series of deletion and point mutants of Sec2p unveiled the catalytic residues for its GEF activity as well as the Sec4p binding site, thus presenting a nucleotide exchange mechanism by a simple coiled coil. The present functional analyses allow us to build the Sec2p:Sec4p complex model, which explains the specificity for Rab GTPases by their respective GEF proteins.