Ferritin protein nanocage ion channels: gating by N-terminal extensions

Ferritin protein nanocages, self-assembled from four-α-helix bundle subunits, use Fe²⁺ and oxygen to synthesize encapsulated, ferric oxide minerals. Ferritin minerals are iron concentrates stored for cell growth. Ferritins are also antioxidants, scavenging Fenton chemistry reactants. Channels for iron entry and exit consist of helical hairpin segments surrounding the 3-fold symmetry axes of the ferritin nanocages. We now report structural differences caused by amino acid substitutions in the Fe²⁺ ion entry and exit channels and at the cytoplasmic pores, from high resolution (1.3–1.8 Å) protein crystal structures of the eukaryotic model ferritin, frog M. Mutations that eliminate conserved ionic or hydrophobic interactions between Arg-72 and Asp-122 and between Leu-110 and Leu-134 increase flexibility in the ion channels, cytoplasmic pores, and/or the N-terminal extensions of the helix bundles. Decreased ion binding in the channels and changes in ordered water are also observed. Protein structural changes coincide with increased Fe²⁺ exit from dissolved, ferric minerals inside ferritin protein cages; Fe²⁺ exit from ferritin cages depends on a complex, surface-limited process to reduce and dissolve the ferric mineral. High concentrations of bovine serum albumin or lysozyme (protein crowders) to mimic the cytoplasm restored Fe²⁺ exit in the variants to wild type. The data suggest that fluctuations in pore structure control gating. The newly identified role of the ferritin subunit N-terminal extensions in gating Fe²⁺ exit from the cytoplasmic pores strengthens the structural and functional analogies between ferritin ion channels in the water-soluble protein assembly and membrane protein ion channels gated by cytoplasmic N-terminal peptides.     

Parenteral immunization of fish,Labeo rohita with Poly d,l-lactide-co-glycolic acid (PLGA) encapsulated antigen microparticles promotes innate and adaptive immune responses

Immunogenicity of different antigen preparations of outer membrane proteins (OMP) of Aeromonas hydrophila such as Poly d, l-lactide-co-glycolic acid (PLGA) microparticles, oil emulsion, neat OMP and bacterial whole cells were compared through intra-peritoneal injection in fish, Labeo rohita. Among these preparations, PLGA encapsulated antigen stimulated both innate and adaptive immune parameters and the immunogenicity exhibited by PLGA microparticles was significantly higher (p < 0.05) at both 21 and 42 days post-immunization suggesting that the above delivery system would be a novel antigen carrier for parenteral immunization in fish, Labeo rohita     

Pyrazoline based MAO inhibitors: synthesis, biological evaluation and SAR studies

Twenty-two pyrazoline derivatives were synthesized and tested for their human MAO (hMAO) inhibitory activity. Twelve molecules with unsubstituted ring A and substituted ring C (5-16) were found to be potent inhibitors of hMAO-A isoform with SI_MAO-A in the order 10³ and 10⁴. Ten molecules with unsubstituted ring A and without ring C (21-30), in which eight molecules (21, 23-26, and 28-30) were selective for hMAO-A, one for hMAO-B (22) and the other one non-selective (27). Presence of ring C increases potency as well as SI towards hMAO-A; however its absence decreases both potency and SI towards hMAO-A and hMAO-B.     

Evaluation of the antimicrobial potential of two flavonoids isolated from limnophila plants

The antimicrobial potential of two bioflavonoids, i.e., 5,7‐dihydroxy‐4′,6,8‐trimethoxyflavone ( 1 ) and 5,6‐dihydroxy‐4′,7,8‐trimethoxyflavone ( 2 ), isolated from Limnophila heterophylla Benth . and L. indica (Linn .) Druce (Scrophulariaceae), respectively, were evaluated against the microbial strains Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Alternaria solani, and Candida albicans. Compounds 1 and 2 exhibited moderate but broad antimicrobial activities against both Gram‐positive and Gram‐negative bacteria and also against the fungal pathogens. Moreover, the mechanism of action of 1 and 2 on the cellular functions or structures of some of the microorganisms was studied. Compound 1 showed a bactericidal effect against E. coli and S. aureus (MICs of 200 and 250 μg/ml, resp.), while compound 2 was found to effectively kill B. subtilis by cell lysis. The growth of A. solani and C. albicans was inhibited by compounds 1 and 2 , respectively. The effects of the flavonoids on the cellular structures and the carbohydrate metabolic pathways were studied by scanning electron microscopy (SEM) of the treated cells and by assessing the specific activity of key enzymes of the pathways, respectively. At sublethal doses, they enhanced the activity of gluconeogenic fructose bisphosphatase, but decreased the activity of phosphofructokinase and isocitrate dehydrogenase, the key enzymes of the Embden Meyerhof Parnas pathway and the tricarboxylic acid cycle, respectively.     

Antigen adsorbed calcium phosphate nanoparticles stimulate both innate and adaptive immune response in fish, Labeo rohita H

Calcium phosphate nanoparticles as an antigen/protein delivery was explored in a fish model Labeo rohita H. S-layer protein (of Aeromonas hydrophila) adsorbed on nano sized calcium phosphate particles elicited both innate and adaptive immune parameters which persisted up to 63 days of post immunization through parenteral immunization and gave cross protections.     

Synthesis, characterization, conformational analysis of a cyclic conjugated octreotate peptide and biological evaluation of 99mTc-HYNIC-His3-Octreotate as novel tracer for the imaging of somatostatin receptor-positive tumors

Peptides are attracting increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. We therefore synthesized new cyclic octapeptides conjugated with HYNIC by Fmoc solid-phase peptide synthesis. These were purified and analyzed by RP-HPLC, MALDI mass, ¹H NMR, ¹³C NMR, HSQC, HMBC, COSY and IR spectroscopy. Conformational analysis of the peptides was performed by circular dichroism spectroscopy, in pure water and trifluoroethanol–water (1:1), revealed the presence of strong secondary structural features like β-sheet and random coils. Labeling was performed with ^99mTc using Tricine and EDDA as coligands by SnCl₂ method to get products with excellent radiochemical purity >99.5 %. Metabolic stability analysis did not show any evidence of breaking of the labeled compounds and formation of free ^99mTc. Internalization studies were done and IC₅₀ values were determined in somatostatin receptor-expressing C6 glioma cell line and rat brain cortex membrane, and the results compared with HYNIC-TOC as standard. The IC₅₀ values of ^99mTc-HYNIC-His³-Octreotate (21 ± 0.93 nM) and ^99mTc-HYNIC-TOC (2.87 ± 0.41 nM) proved to be comparable. Biodistribution and image study on normal rat under gamma camera showed very high uptake in kidney and urine, indicating kidney as primary organ for metabolism and route of excretion. Biodistribution and image study on rats bearing C6 glioma tumor found high uptake in tumor (1.27 ± 0.15) and pancreas (1.71 ± 0.03). Using these findings, new derivatives can be prepared to develop ^99mTc radiopharmaceuticals for imaging somatostatin receptor-positive tumors.     

Role of sedoheptulose-1,7 bisphosphatase in low light tolerance of rice (Oryza sativa L.)

Rice grain yield is drastically reduced under low light especially in kharif (wet) season due to cloudy weather during most part of crop growth. Therefore, 50–60% of yield penalty was observed. To overcome this problem, identification of low light tolerant rice genotypes with a high buffering capacity trait such as photosynthetic rate has to be developed. Sedoheptulose-1,7 bisphosphatase, a light-regulated enzyme, plays pivotal role in the Calvin cycle by regenerating the substrate (RuBP) for RuBisCo and therefore, indirectly regulates the influx of CO₂ for this crucial process. We found a potential role of SBPase expression and activity in low light tolerant and susceptible rice genotypes by analyzing its influence on net photosynthetic rate and biomass. We observed a significant relationship of yield with photosynthesis, SBPase expression and activity especially under low light conditions. Two tolerant and two susceptible rice genotypes were used for the present study. Tolerant genotypes exhibited significant but least reduction compared to susceptible genotypes in the expression and activity of SBPase, which was also manifested in its photosynthetic rate and finally in the grain yield under low light. However, susceptible genotypes showed significant reduction in SBPase activity along with photosynthesis and grain yield suggesting that tracking the expression and activity of SBPase could form a simple and reliable method to identify the low light tolerant rice cultivars. The data were analyzed using the Indostat 7.5, Tukey–Kramer method through Microsoft Excel 2019 and PAST4.0 software. The significant association of SBPase activity with the grain yield, net assimilation rate, electron transfer rate, biomass and grain weight were observed under low light stress. These traits should be considered while selecting and breeding for low light tolerant cultivars. Thus, SBPase plays a major role in the low light tolerance mechanism in rice.Electronic supplementary materialThe online version of this article (10.1007/s12298-020-00905-z) contains supplementary material, which is available to authorized users.     

Identification of a TLR-independent pathway for Borrelia burgdorferi-induced expression of matrix metalloproteinases and inflammatory mediators through binding to integrin alpha 3 beta 1

Borrelia burgdorferi stimulates a robust inflammatory response at sites of localization. Binding of borrelial lipoproteins to TLR-2 is one pathway important in the host response to B. burgdorferi. However, while TLR-2 is clearly important in control of infection, inflammation is actually worsened in the absence of TLR-2 or the shared TLR adapter molecule, MyD88, suggesting that there are alternative pathways regulating inflammation. Integrins are cell surface receptors that play an important role in cell to cell communications and that can activate inflammatory signaling pathways. In this study, we report for the first time that B. burgdorferi binds to integrin alpha(3)beta(1) and that binding of B. burgdorferi to this integrin results in induction of proinflammatory cytokines, chemokines, and end-effector molecules such as matrix metalloproteinases in primary human chondrocyte cells. Expression of these same molecules is not affected by the absence of MyD88 in murine articular cartilage, suggesting that the two pathways act independently in activating host inflammatory responses to B. burgdorferi. B. burgdorferi-induced alpha(3) signaling is mediated by JNK, but not p38 MAPK. In summary, we have identified a new host receptor for B. burgdorferi, integrin alpha(3)beta(1); binding of B. burgdorferi to integrin alpha(3)beta(1) results in the release of inflammatory mediators and is proposed as a TLR-independent pathway for activation of the innate immune response by the organism.     

Differential cytokine mRNA expression in Dermatophagoides farinae allergen-sensitized and respiratory syncytial virus-infected mice

The interaction between mite allergen sensitization and respiratory syncytial virus (RSV) infection at the level of cytokine mRNA expression was examined in a murine model in the present study. Primary RSV infection enhances expression of inflammatory cytokines such as IL-6, IFN-gamma, and eotaxin in the lung and upregulates the expression of Th2-like cytokines IL-10 and IL-13 in the spleen in BALB/c mice. Mite antigen-sensitized and RSV-infected (ASRSV) mice show enhanced (P < 0.05) total serum IgE compared to antigen-sensitized mice. However, the levels of viral mRNA in the lung tissues are comparable between RSV-infected and ASRSV mice. It is concluded that compartmentalization of cytokine expression following RSV infection plays a role in the augmentation of Th2-like and IgE antibody response to RSV.