How the Nucleus and Mitochondria Communicate in Energy Production During Stress: Nuclear MtATP6, an Early-Stress Responsive Gene, Regulates the Mitochondrial F1F0-ATP Synthase Complex

A small number of stress-responsive genes, such as those of the mitochondrial F₁F₀-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F₀ part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F₁F₀-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.     

Accounting for robustness in modeling signal transduction responses

Abstract

A classical question in systems biology is to find a Boolean model which is able to predict the observed responses of a signaling network. It has been previously shown that such models can be tailored based on experimental data. While fitting a minimum-size network to the experimentally observed data is a natural assumption, it can potentially result in a network which is not so robust against the noises in the training dataset. Indeed, it is widely accepted now that biological systems are generally evolved to be very robust. Therefore, in the present work, we extended the classical formulation of Boolean network construction in order to put weight on the robustness of the created network. We show that our method results generally in more relevant networks. Consequently, considering robustness as a design principle of biological networks can result in more realistic models.     

Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography

Abstract

The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400?SEC, we obtained highly pure, i.e.,?>?90%, rHBsAg virus-like particles (VLP).     

Molecular detection and genotyping of Fusarium oxysporum f. sp. psidii isolates from different agro-ecological regions of India

Twenty one isolates of Fusarium oxysporum f. sp. psidii (Fop), causing a vascular wilt in guava (Psidium guajava L.), were collected from different agro-ecological regions of India. The pathogenicity test was performed in guava seedlings, where the Fop isolates were found to be highly pathogenic. All 21 isolates were confirmed as F. oxysporum f. sp. psidii by a newly developed, species-specific primer against the conserved regions of 28S rDNA and the intergenic spacer region. RAPD and PCR-RFLP were used for genotyping the isolates to determine their genetic relationships. Fifteen RAPD primers were tested, of which five primers produced prominent, polymorphic, and reproducible bands. RAPD yielded an average of 6.5 polymorphic bands per primer, with the amplified DNA fragments ranging from 200–2,000 bp in size. A dendrogram constructed from these data indicated a 22–74% level of homology. In RFLP analysis, two major bands (350 and 220 bp) were commonly present in all isolates of F. oxysporum. These findings provide new insight for rapid, specific, and sensitive disease diagnosis. However, genotyping could be useful in strain-level discrimination of isolates from different agro-ecological regions of India.     

In vitro cytotoxic,antibacterial, antifungal and urease inhibitory activities of some N4- substituted isatin-3-thiosemicarbazones

A series of 15 previously reported N⁴-substituted isatin-3-thiosemicarbazones 3a-o has been screened for cytotoxic, antibacterial, antifungal and urease inhibitory activities. Compounds 3b, 3e and 3n proved to be active in cytotoxicity assay; 3e exhibited a high degree of cytotoxic activity (LD₅₀ = 1.10 × 10^{? 5} M). Compound 3h exhibited significant antibacterial activity against B. subtilis, whereas compounds 3a, 3k and 3l displayed significant antifungal activity against one or more fungal strains i.e. T. longifusus, A. flavus and M. canis. In human urease enzyme inhibition assay, compounds 3g, 3k and 3m proved to be the most potent inhibitors, exhibiting relatively pronounced inhibition of the enzyme. These compounds, being non-toxic, could be potential candidates for orally effective therapeutic agents to treat certain clinical conditions induced by bacterial ureases like H. pylori urease. This study presents the first example of inhibition of urease by isatin-thiosemicarbazones and as such provides a solid basis for further research on such compounds to develop more potent inhibitors.     

Dual Substrate Model for Novel Approach Towards a Kinetic Study of Acetylcholinesterase Inhibition by Diazinon

Limited reports as compared to other insecticides appear in the literature for acetylcholinester-ase (AChE) inhibition by diazinon. In the current study, new kinetic parameters of AChE inhibition by diazinon have been investigated. The assay was done with bovine retinal AChE using two different substrate (ASCh) concentrations in the absence and presence of diazinon (0.08-1.28 mM). The optical density was monitored up to 25min (reaction time) for the assay. New kinetic parameters (k¹_oms, k¹_sms, k¹_oms, k¹_sms, k¹_asms and k¹_asms) were calculated from these experimental data.     

Genotyping of –374A/T, –429A/G,And 63 bp Ins/Del Polymorphisms of Rage By Rapid One-Step Hexaprimer Amplification Refractory Mutation System Polymerase Chain Reaction in Breast Cancer Patients

Several studies have focused on the RAGE genetic background and have demonstrated that its polymorphisms affect the receptor's activity, expression, and downstream signaling. However, there is only little information regarding RAGE polymorphism in breast cancer. In the present study, the authors studied RAGE polymorphisms in 71 patients with breast cancer and 93 healthy women. RAGE –374T/A, –429T/C, and 63 bp Ins/del polymorphisms were analyzed using a hexaprimer amplification refractory mutation system PCR (H-ARMS-PCR). The results showed that RAGE polymorphisms are not associated with breast cancer in the current study population. Larger studies are required to confirm these data in other populations.     

When to estimate sex ratio in natural populations of insects? A study on sex ratio variations of gall midges within a generation

Precise estimation of arthropods' sex ratio is an important issue in a wide range of ecological studies and biological control programs. Although, in many cases changes in arthropods' sex ratio may be under the control of parents or some symbiotic microorganisms, biased sex ratios in some other species are caused by some extrinsic factors, neglect of which may lead to under/overestimation of true sex ratio. In this paper, we pursued those factors that cause false estimation of sex ratio in insects' species. We studied the predatory gall midge, Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae), an important biological control agent of aphids, that shows protandry (i.e. early male emergence), differential lifespan of sexes, and differential distribution of sexes across habitat. Ten populations of A. aphidimyza were released separately in transparent cages and their sex ratio variations were recorded every 12 hours. The primary sex ratio in this species seems to be slightly male‐biased (52.41% males), however early emergence of males biases the sex ratio up to 72% males in a few hours after emergence. Shortly after the emergence of females, the sex ratio reaches its primary situation, but as a result of male‐biased mortality after mating, the proportion of females increases gradually to 97% by the fourth and fifth days after emergence. These results explicitly suggest that direct estimation of sex ratio in natural populations may be affected by some secondary factors such as differential mortality of sexes, protandry, and differential distribution of males and females over time and/or across habitat.     

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10⁴ IU mg^?1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.