Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19

Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water–gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B₁₂ (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B₁₂ was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.     

Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ_RpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZ_RpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZ_RpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n?=?2–6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.     

A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences

Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.     

Amelioration of cognitive impairment and neurodegeneration by catechin hydrate in rat model of streptozotocin-induced experimental dementia of Alzheimer’s type

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder resulting in cognitive decline and enhancement of oxidative loads in the brain. Flavonoids have been considered to exert human health benefits by anti-oxidant and anti-inflammatory properties. The present study is aimed to elucidate the neuroprotective effect of catechin hydrate (CH), a natural flavanoid with potential antioxidant and anti-inflammatory properties, on intracerebroventricular streptozotocin (ICV-STZ) induced neuronal loss and memory impairment. To test this hypothesis, male Wistar rats were pretreated with CH (10 and 20 mg/kg bwt) orally once daily for 21 days and then bilaterally injected with ICV-STZ (3 mg/kg bwt), while sham group rats receive the same volume of vehicle. After 2 weeks of ICV-STZ infusion, rats were tested for cognitive performance using Morris water maze (MWM) test and then sacrifice for biochemical and histopathological assays. CH was found to be successful in upregulating the antioxidant status and prevented the memory loss. The expression of choline acetyl transferase (ChAT) was decreased in ICV-STZ group and CH pretreatment increases the expression of ChAT. Moreover, inflammatory mediators like TNF-α, IL-1β levels and expression of iNOS were significantly attenuated by CH pretreatment. The study suggests that CH is effective in preventing memory loss, ameliorating the oxidative stress and might be beneficial for the treatment of sporadic dementia of Alzheimer’s type (SDAT).     

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC₅₀ ∼ 20.7 μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC₅₀ ∼ 7.2 μM) as compared to diospyrin (IC₅₀ ∼ 12.6 μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC₅₀ ∼ 0.18 μM). Also, treatment of infected BALB/c mice with D17 at 2 mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

Spinigerin induces apoptotic like cell death in a caspase independent manner in Leishmania donovani

Antimicrobial peptides (AMPs) are multifunctional components of the innate immune system. Chemotherapeutic agents used for treatment of visceral leishmaniasis (VL) are now threatened due to the emergence of acquired drug resistance and toxicity. AMPs are attractive alternative to conventional pharmaceuticals. In this study, first time we explored the antileishmanial activity of spinigerin originally derived from Pseudacanthotermes spiniger. Leishmania donovani promastigotes present apoptosis-like cell death upon exposure to spinigerin (IC₅₀, 150 μM). The infection rate was reduced by 20% upon exposure to 150 μM spinigerin but no cytotoxicity on host macrophages was observed. Elevation of intracellular ROS level and down-regulation of two ROS detoxifying enzymes, ascorbate peroxidase (APx) and trypanothione reductase (TR) suggested essential role of ROS machinery during spinigerin mediated cell death. About 97% cell population was found to be Annexin-V positive; 44% cells being highly Annexin-V positive. Moreover, we observed morphological changes like cell rounding, nuclear condensation, oligonucleosomal DNA degradation and TUNEL positive cells without loss of membrane integrity upon spinigerin exposure, suggests apoptosis-like death. Interestingly, collapse in mitochondrial membrane potential and increased level of intracellular ROS and calcium were not associated with caspase like activity. Computational analysis suggests spiningerin interacts with trypanothione reductase and thus probably interferes its function to detoxify the toxic ROS level. Therefore, spinigerin induces apoptosis-like cell death in L. donovani in a caspase-independent manner. The study elucidates the antileishmanial property of spinigerin that may be considered for future chemotherapeutic option alone or adjunct with other drug regimens for improved treatment of visceral leishmaniasis.     

Pressure and Temperature Dependence of Growth and Morphology of Escherichia coli: Experiments and Stochastic Model

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1–400 atm) and temperature (23–40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.