Metal migration and subunit swapping in ALS-linked SOD1: Zn2+ transfer between mutant and wild-type occurs faster than the rate of heterodimerization

The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔG_Het) between WT and ALS-mutant SOD1 in mismatched metalation states—where one subunit is metalated and the other is not—have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by “stealing” metal ions remains untested. This study used capillary zone electrophoresis and mass spectrometry to track heterodimerization and metal transfer between WT SOD1, ALS-variant SOD1 (E100K, E100G, D90A), and triply deamidated SOD1 (modeled with N26D/N131D/N139D substitutions). We determined that rates of subunit exchange between apo dimers and metalated dimers—expressed as time to reach 30% heterodimer—ranged from t_30% = 67.75 ± 9.08 to 338.53 ± 26.95 min; free energies of heterodimerization ranged from ΔG_Het = -1.21 ± 0.31 to -3.06 ± 0.12 kJ/mol. Rates and ΔG_Het values of partially metalated heterodimers were more similar to those of fully metalated heterodimers than apo heterodimers, and largely independent of which subunit (mutant or WT) was metal-replete or metal-free. Mass spectrometry and capillary electrophoresis demonstrated that mutant or WT 4Zn-SOD1 could transfer up to two equivalents of Zn²⁺ to mutant or WT apo-SOD1 (at rates faster than the rate of heterodimerization). This result suggests that zinc-replete SOD1 can function as a chaperone to deliver Zn²⁺ to apo-SOD1, and that WT apo-SOD1 might increase the toxicity of mutant SOD1 by stealing its Zn²⁺.     

Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli

HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function.     

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.     

RNA-seq analysis reveals a key role of brassinolide-regulated pathways in NaCl-stressed cotton

Brassinolide (BL) alleviates salt injury in cotton seedlings; however, little is known about the molecular mechanisms of this response. In this study, digital gene expression analysis was performed to better understand the regulatory pathways of BL in NaCl-stressed cotton (Gossypium hirsutum L.). Compared with control plants (CK), a total of 1 162 and 7 659 differentially expressed genes (DEGs) were detected in the leaves and roots of NaCl-treated plants, respectively. Most of the DEGs in NaCl-treated plants, compared to CK, were regulated by BL. Moreover, expression patterns of DEGs in BL+NaCl treated plants were similar to those in CK plants; however, the responses of DEGs in the leaves and roots of NaCl-treated plants to BL differed. In the roots, BL-regulated DEGs were involved in protein biosynthesis, whereas in the leaves, BL promoted photosynthesis in NaCl-stressed cotton. BL treatment also significantly increased the overall biomass, chlorophyll a + b content in leaves, and the protein content in roots in NaCl-stressed cotton. The downregulation of stress-responsive genes in BL+NaCl-stressed leaves was also found. These results suggest that BL can alleviate NaCl injury in cotton plants.     

Synchronized oviposition triggered by migratory flight intensifies larval outbreaks of beet webworm

Identifying the reproductive consequences of insect migration is critical to understanding its ecological and evolutionary significance. However, many empirical studies are seemingly contradictory, making recognition of unifying themes elusive and controversial. The beet webworm, Loxostege sticticalis L. is a long-range migratory pest of many crops in the northern temperate zone from 36 °N to 55 °N, with larval populations often exploding in regions receiving immigrants. In laboratory experiments, we examined (i) the reproductive costs of migratory flight by tethered flight, and (ii) the reproductive traits contributing to larval outbreaks of immigrant populations. Our results suggest that the beet webworm does not initiate migratory flight until the 2nd or 3rd night after emergence. Preoviposition period, lifetime fecundity, mating capacity, and egg hatch rate for adults that experienced prolonged flight after the 2nd night did not differ significantly from unflown moths, suggesting these traits are irrelevant to the severity of beet webworm outbreaks after migration. However, the period of first oviposition, a novel parameter developed in this paper measuring synchrony of first egg-laying by cohorts of post-migratory females, for moths flown on d 3 and 5 of adulthood was shorter than that of unflown moths, indicating a tightened time-window for onset of oviposition after migration. The resulting synchrony of egg-laying will serve to increase egg and subsequent larval densities. A dense population offers potential selective advantages to the individual larvae comprising it, whereas the effect from the human standpoint is intensification of damage by an outbreak population. The strategy of synchronized oviposition may be common in other migratory insect pests, such as locust and armyworm species, and warrants further study.     

Intentionally introduced species: more easily invited than removed

Alien species are brought into countries world wide on a massive scale for agricultural production, ex situ conservation, landscape aesthetics, gardens, and ecosystem restoration. Unfortunately, some of these species have escaped and adversely impacted on regional as well as global biodiversity conservation and agricultural production. To reduce such risks, it is necessary to implement specific and effective measures. Since various government departments and institutions are involved in the management of alien species, it is difficult to prevent native and agroecosystems from being invaded by invited species. We propose the establishment of a supervision and inspection continuum over intentional species introduction, similar to that which exists in some countries over unintentional species introductions. Namely, a justification of the necessity to import, a risk assessment, assurances as to provision of an adequate containment facility assessment, and a damage-limitation protocol should that need to be invoked. These requirements should be satisfied before an alien species is knowingly imported, and the necessary follow-up supervision is important post- importation.