Cold Acclimation in Arabidopsis thaliana

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT₅₀) of about −3°C to an LT₅₀ of about −6°C after 24 hours at 4°C; LT₅₀ values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A⁺) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.     

Proteolysis to Identify Protease Substrates: Cleave to Decipher

Proteolysis is an irreversible post‐translational modification process, characterized by highly precise yet stable cleavage of proteins. Downstream events in signaling processes are reliant on proteolysis triggered by the protease activity. Studies indicate that abnormal proteolytic activity may lead to the manifestation of diseased conditions. Therefore, characterization of proteases may provide clues to understand their role in fundamental cellular processes like cellular growth, differentiation, apoptosis, and survival. The relevance of proteases and their substrates as clinical targets are being studied. Understanding the mechanism of proteolytic activity, the identity, and the role of repertoire of its substrates in a physiological pathway has opened avenues for novel drug designing. However, only a limited knowledge of protease substrates is currently available. In this review, the authors recapitulate the library screening, proteomics, and bioinformatics based approaches that have been employed for the identification of protease substrates.     

Ca uptake and plasma membrane depolarization associated with blue light-sensitive exogenous NADH oxidation by Cuscuta protoplasts

Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca²⁺-uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca²⁺ uptake by the protoplasts both in dark and under blue light. The Ca²⁺-calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca²⁺-uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca²⁺-calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta     

Mutations in CSPP1 Lead to Classical Joubert Syndrome

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia.     

Neonatal amygdalae and hippocampi are influenced by genotype and prenatal environment,and reflected in the neonatal DNA methylome

The amygdala and hippocampus undergo rapid development in early life. The relative contribution of genetic and environmental factors to the establishment of their developmental trajectories has yet to be examined. We performed imaging on neonates and examined how the observed variation in volume and microstructure of the amygdala and hippocampus varied by genotype, and compared with prenatal maternal mental health and socioeconomic status. Gene × Environment models outcompeted models containing genotype or environment only to best explain the majority of measures but some, especially of the amygdaloid microstructure, were best explained by genotype only. Models including DNA methylation measured in the neonate umbilical cords outcompeted the Gene and Gene × Environment models for the majority of amygdaloid measures and minority of hippocampal measures. This study identified brain region‐specific gene networks associated with individual differences in fetal brain development. In particular, genetic and epigenetic variation within CUX1 was highlighted.     

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.