The C‐terminal calcium‐sensitive disordered motifs regulate isoform‐specific polymerization characteristics of calsequestrin

Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C‐terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn‐motif and DEXn‐motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C‐terminal motifs possess Ca²⁺‐sensitivity and affect protein function. Sequence analysis shows that both the Dn‐ and DEXn‐motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca²⁺‐mediated folding suggesting that these disordered motifs are Ca²⁺‐sensitivity. We generated chimeric proteins by swapping the C‐terminal portions between CASQ1 and CASQ2. Our studies show that the C‐terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C‐terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca²⁺‐sensitivity IDRs located at the back‐to‐back dimer interface influence isoform‐specific Ca²⁺‐dependent polymerization properties of CASQ. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 15–22, 2015.     

Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants

CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.     

Glypican 4 mediates Wnt transport between germ layers via signaling filopodia

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4–labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Mitofusin 1 and optic atrophy 1 shift metabolism to mitochondrial respiration during aging

Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re‐enter the proliferative cycle. This study investigated whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were taken in quiescent NHFs from younger (newborn, 3‐day, 5‐month, and 1‐year) and older (58‐, 61‐, 63‐, 68‐, and 70‐year) healthy donors as well as NHFs from the same individual at different ages (29, 36, and 46 years). Results show significant changes in cellular metabolism during aging of quiescent NHFs: Old NHFs exhibit a significant decrease in glycolytic flux and lactate levels, and increase in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from the Seahorse XF Cell Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more toward fusion. Genetic knockdown of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more toward glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation‐induced increase in doubling time of NHFs. In summary, results show that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during chronological lifespan, and MFN1 and OPA1 regulate this process.     

Growth promotion and induced disease suppression of four vegetable crops by a selected plant growth-promoting rhizobacteria (PGPR) strain Bacillus subtilis 21-1 under two different soil conditions

This study aimed at investigating the efficacy of a PGPR strain, Bacillus subtilis 21-1 (BS21-1), under two different soil conditions for plant growth promotion and disease suppression. BS21-1 treatment significantly (P < 0.05) promoted plant growth as measured by plant height and leaf width and increased the seed germination rate in organic soil (OS) compared with seed bed soil (SBS). In Chinese cabbage and lettuce, soft rot disease was reduced to 45 and 23.5 %, respectively, by BS21-1, and to 33 and 52.5 %, respectively, by benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester (BTH) treatments in OS compared with SBS. These levels of disease suppression were greater than those found in the water-treated control upon pathogen challenge. There was a greater reduction of anthracnose lesions on the leaves of cucumber plants treated with BS21-1 and BTH in OS when compared with SBS. Botrytis rot disease in tomato caused by Botrytis cinerea was drastically reduced to 2 % in OS and 4 % in SBS by BS21-1 treatment. In the four plants studied, there was increased disease suppression in OS compared with SBS. Upon treatment with BS21-1, there was an increased expression of the PR-1a gene by β-gulcuronidase (GUS) activity in tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants in OS compared with SBS, which indicates a possible role of the SA pathway in BS21-1 mediated plant protection. Thus, the isolate BS21-1 could effectively be used as one of the biocontrol agents for disease suppression in four vegetable crops through systemic resistance and for plant growth promotion.     

New diterpenoids from Caesalpinia species and their cytotoxic activity

Chemical investigation on Caesalpinia crista afforded two new diterpenoids, 6β-cinnamoyloxy-7β-acetoxyvouacapen-5α-ol and 6β,7β-dibenzoyloxyvouacapen-5α-ol and on Caesalpinia pulcherrima another new diterpenoid, 12-demethyl neocaesalpin F along with several known constituents. The structures of the new compounds were settled from their 1D and 2D NMR spectral data. The cytotoxicity of these compounds was measured on two different cancer cell lines.     

Induced suppression of soft rot disease in tobacco by combined application of Bacillus subtilis strain B4 and chemical elicitor BTH

In this study, the utility of the combined application of Benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester (BTH), a systemic acquired resistance (SAR) inducer, and the plant growth-promoting rhizobacteria (PGPR), Bacillus subtilis strain B4 (B4), was investigated for the suppression of soft rot disease caused by Pectobacterium carotovorum SCC1 (SCC1) in tobacco seedlings. Soft rot disease was completely suppressed in tobacco with combined application of B4 strain and 0.1 mM BTH when compared to individual application upon pathogen challenge. In addition, the population of bacterial cells of B4 strain was found to be greater when cultured in growth media in the presence of BTH, compared to the growth of B4 strain in the absence of BTH. Spectrophotometer analysis revealed that there was an increased broad range of compounds in the culture filtrate of B4 strain when grown with BTH, compared to the culture filtrate of B4 strain alone. The combined application of B4 strain and 0.1 mM BTH induced the increased expression of PR1a::GUS on tobacco and elicited systemic resistance against SCC1 when compared to individual application. However, there was low expression of PR1a::GUS in the water-treated control. Hence, the integrated use of BTH and B4 strain might be one of the strategies for biological control of soft rot disease through induced systemic resistance (ISR) in tobacco plants.     

New bioactive macrocyclic diterpenoids from Jatropha multifida

Two new macrocyclic diterpenoids, multifidanol (1) and multifidenol (2) along with several known compounds have been isolated from the stem of Jatropha multifida. The structures of the new compounds were established from the extensive studies of their 1D and 2D NMR spectra. The cytotoxic and antimicrobial activities of these two constituents were examined.     

Multifidone: A novel cytotoxic lathyrane-type diterpene having an unusual six-membered A ring from Jatropha multifida

Chemical examination of the stems of Jatropha multifida afforded a novel lathyrane-type diterpene, multifidone, having an unusual six-membered A ring. The structure of the compound was determined from detailed analysis of its 1D and 2D NMR spectra and X-ray crystallographic analysis. Its cytotoxicity was measured on four different cancerous cell lines.