The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.     

Growth factors upregulate deposition and remodeling of ECM by endothelial cells cultured for tissue-engineering applications

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial for their long-term function. The interaction between cells and extra-cellular components is indispensable in determining cellular behavior in tissues and on biomaterials. The fibrin that contains fibronectin shows promise in most aspects as a tissue engineering scaffold, whereas, deposition of elastin and collagen by endothelial cells grown in the lumen of the construct is desirable to improve post implant retention, mechanical stability and vaso-responsiveness. So far there is no report on production of extra-cellular matrix (ECM) proteins, elastin and collagen by endothelial cells (EC) in in vitro culture conditions. In this study, we have used a biomimetic approach of providing multiple growth factors (GF) in the fibronectin (FN)-containing fibrin matrix to induce production of elastin and collagen by the endothelial cells for application in vascular tissue engineering. Deposition of elastin and collagens with matrix remodeling is demonstrated through qualitative analysis of the matrices that were recovered after growing cells on the initial fibrin-FN-GF matrix. Expressions of mRNA for both proteins were assessed by real time polymerase chain reaction (RT-PCR) to estimate the effects of multiple growth factor compositions. Marked deposition of elastin and collagen was evidenced by staining the recovered matrix after different culture intervals. Obviously, the biomimetic environment created by adding angiogenic and platelet growth factors in the fibrin-fibronectin-gelatin matrix can induce deposition of collagens and elastin by EC.     

Determination and quantification of asiaticoside in endophytic fungus from <Emphasis Type="Italic">Centella asiatica</Emphasis> (L.) Urban

Centella asiatica (L.) Urban is a highly considered medicinal plant owing to its secondary metabolites asiaticoside, madecassoside, asiatic acid, and madecassic acid. The asiaticoside, one of the most important constituents of the plant, is a triterpenoid saponin having memory enhancement property. Given its medicinal properties, we isolated and characterized endophytic fungi from this plant with the aim to screen these microorganisms for asiaticoside production. In total, we isolated 13 endophytic fungi from the leaves of the plant, out of which one of the isolates produced asiaticoside. This asiaticoside producing isolate was identified as Colletotrichum gloeosporioides by internal transcribed spacer-based rDNA sequencing. The presence of asiaticoside in ethyl acetate extract of C. gloeosporioides was confirmed by LC–MS. The production of asiaticoside measured in relation to incubation time and subculture generation revealed presence of 62.29?±?3.36 µg/100 mL of asiaticoside by C. gloeosporioides on the 15th day in first subculture generation followed by a decrease in subsequent generations. A similar trend was also shown by yield and growth curve of C. gloeosporioides. The asiaticoside production and yield were found to be positively correlated. This paper reported the production of asiaticoside by an endophytic fungus C. gloeosporioides for the first time. The present findings definitely provide an impetus to the production of asiaticoside by utilizing the endophytic source.

Graphical Abstract

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Chemical compound studied in this article: Asiaticoside (PubChemCID: 108062)

     

Morphological,phytochemical and genetic diversity of threatened Polygonatum verticillatum (L.) All. populations of different altitudes and habitat types in Himalayan region

Polygonatum verticillatum (L.) All. is an important medicinal herb that belongs to the family Asparagaceae. The rhizome of the species is used in Chyavanprash preparation and several other ayurvedic formulations. Numerous active constituents like saponins, alkaloids, phytohormones, flavonoids, antioxidants, lysine, serine, aspartic acid, diosgenin, β-sitosterol, etc. have been reported from this species. In this study, morphological, phytochemical, antioxidant and genetic variations of 11 distant populations of P. verticillatum were measured. Considerably (P < 0.05) higher variations were recorded among different populations of P. verticillatum using morphological, phytochemical and genetic diversity parameters. AGFW (above ground fresh weights); flavonols, FRAP (Ferric ion reducing antioxidant power) and NO (Nitric Oxide scavenging activity) were recorded maximum in Kafni population. Similarly, a significantly higher above and below ground dry weight was recorded in Mayawati and Surmoli populations respectively. Maximum phenolic content, tannins, and DPPH (2,2-diphenyl-1-picrylhydrazyl) activity were recorded in Milam population. A total of 165 individuals from 11 populations were assessed for genetic diversity using inter-simple sequence repeats (ISSR) marker. High genetic diversity (He = 0.35) was recorded in Himkhola and Surmoli populations while it was observed minimum (0.28) in the Mayawati population. Altitude showed a significant positive correlation with tannins (r = 0.674; P < 005) and DPPH (r = 0.820; P < 0.01). Phenol content exhibited a considerably positive relationship with He (r = 0.606; P < 0.05) and BGFW (r = 0.620; P < 0.05), flavonol displayed a positive correlation with Pp% (r = 0.606; P < 0.05). The population structure of P. verticillatum, exhibited that the optimal value of the K was 3 for its populations as determined by the ΔK statistic structure. Among populations, the amount of gene flow is higher (Nm = 1.717) among all sites. Hence, it can be concluded that P. verticillatum populations possess considerable variability in the collected populations. Likewise, the populations from Kafni, Satbunga and Himkhola with higher morphological, phytochemicals and genetic variability were prioritized and therefore recommended for cultivation and mass multiplication to meet the industrial demand for target species.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01044-9.     

CsrA and TnaB coregulate tryptophanase activity to promote exotoxin-induced killing of Caenorhabditis elegans by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli(EPEC) requires the tnaA-encoded enzyme tryptophanase and its substrate tryptophan to synthesize diffusible exotoxins that kill the nematode Caenorhabditis elegans. Here, we demonstrate that the RNA-binding protein CsrA and the tryptophan permease TnaB coregulate tryptophanase activity, through mutually exclusive pathways, to stimulate toxin-mediated paralysis and killing of C. elegans.     

Transmission of experimentally-induced rat virus infection

The duration of transmission of rat virus (RV) infection was determined using Sprague-Dawley rats inoculated oronasally as juveniles (4 weeks old) or as infants (2 days old). Contact transmission from rats inoculated as juveniles was detected for 3 weeks, whereas transmission from rats inoculated as infants occurred for 10 weeks. Transmission continued for at least 7 weeks after seroconversion occurred in rats inoculated as infants. Two of three rats that had ceased to transmit infection harbored infectious virus as detected by explantation of kidney. Intrauterine transmission occurred only after pregnant dams were inoculated with large doses of virus and was more efficient when virus was inoculated intravenously than by the oronasal route. Enzyme immunoassay antibody titers to RV in offspring of previously infected dams decreased steadily during the first 13 weeks of life and 27 of 29 offspring tested by immunofluorescence assay at 12 or 13 weeks of age were seronegative. These results indicate that RV was transmitted by rats inoculated as infants for long periods after seroconversion occurred. They also suggest that the offspring of previously-infected dams were not infected. In utero transmission of RV-Y is unlikely to occur after oronasal inoculation unless rats are exposed to large doses of virus.     

On the macro-micro-morphology of organs of host invasion in hemiparasite Helicanthes elasticus (Desv.) Danser

Loranthaceae family includes hemiparasitic members which are seen invading a wide range of commercial crops. Helicanthes elasticus (Desv.) Danser is very common on mango trees. Though parasitic in nature, this mistletoe is also medicinally important as fetoprotective, against vesicular calculi and kidney infections. This study is an attempt to document macro-microscopical features of parasitic root, fruit and host-mistletoe tissue interaction in the haustorium of H. elasticus growing on mango stems. Collection, preservation, sectioning, staining and photomicrography of the root, fruit and host-mistletoe union were done as per standard methodologies of anatomical studies. Though there is resemblance to the normal roots in morphology as well as anatomy, the microscopic finding of large number of branched stone cells in the roots is interesting. The morpho-anatomical features recorded would help in understanding the infection biology of this mistletoe. The eradication during the earlier stages of its establishment from seed or from the root creeping over the surface of the host can help in controlling this parasite infection on commercially important host plants.     

Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive ³H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive ³H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive ³H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.     

Minocycline,levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease

Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes.