Simultaneous saccharification and protein enrichment fermentation of sugar beet pulp

Summary A product with 40 % protein content was obtained from sugar beet pulp (1.25–2.0 mm) in 48 h one stage (simultaneous) saccharification/fermentation process under optimized conditions using a specific enzyme mixture andCandida tropicalis strain, also saving about 40 % enzymes in comparison to a 2-stage process.     

Selection of preculture conditions for solid state fermentation of sugar beet pulp

Summary For the protein upgrading of sugar-beet pulp in solid state fermentation byTrichoderma reesei andFusarium oxysporum, serveral conditions were studied to prepare an economical preculture for large scale process. The best performance was shown by a preculture obtained in 24 h from 1.5 % molasses solution at pH 4.5–5.0 with 1.0 % milled beet pulp.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹ and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β-branched D -aliphatic residues at position 7 combined with bulky L -aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D -Arg⁰, Hyp³, Thi⁵, D -Cpg⁷, Cpg⁸]-BK [Cpg: α-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L -proline; Thi: β-(2-thienyl)-L -alanine] ( I ), [D -Arg⁰, Hyp³, D -Cpg⁷, Cpg⁸] -BK ( II ), as well as its variant with D -Cpg⁷ replaced by Cpg⁷, namely [D -Arg⁰, Hyp³, Cpg⁷, Cpg⁸]-BK ( III ). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II , in direct correlation with the peptide activities. No turn-like structure was found for residues 6–9. In peptide III , a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists ( I , II ) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D -Cpg⁷]-BK ( IV ) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.     

Use of the Reverse Transcription-Polymerase Chain Reaction for the Detection of Pelargonium Flower Break Carmovirus

A polymerase chain reaction (PCR)-based assay was used to detect pelargonium flower break carmovirus (PFBV) in total RNA extractions made from infected Pelargonium plants. Extracts were reverse transcribed (RT) and the resultant cDNA was amplified by PCR, using oligonucleotide primers specific for 343, 510 and 832 base pair fragments of the RNA-dependent RNA polymerase gene of PFBV.
The specificity and sensitivity of RT-PCR were compared with the enzyme-linked immunosorbent assay (ELISA) for the detection of PFBV in Pelargonium tissues. The virus could be detected efficiently in high dilutions of sap from infected plants and at low concentrations of purified virus. Although ELISA is a powerful tool for virus detection, RT-PCR was over 1000 times more sensitive in detecting PFBV in leaf extracts of infected Pelargonium than was ELISA. The limit of detecting PFBV RNA by RT-PCR was 200 fg, compared with 200 pg of virus by ELISA.     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Ubiquitin‐Activated Interaction Traps (UBAITs) identify E3 ligase binding partners

We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ub iquitin‐A ctivated I nteraction T raps) are E3‐ubiquitin fusion proteins and, in an E1‐ and E2‐dependent manner, the C‐terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co‐purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester‐linked lariat intermediate or through an E2 thioester intermediate, and both WT and active‐site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double‐strand break repair. Using the RNF168 UBAIT, we identify H2AZ—a histone protein involved in DNA repair—as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.     

Emerging Molecular Tools for Engineering Phytomicrobiome

Microbial plant interaction plays a major role in the sustainability of plants. The understanding of phytomicrobiome interactions enables the gene-editing tools for the construction of the microbial consortia. In this interaction, microbes share several common secondary metabolites and terpenoid metabolic pathways with their host plants that ensure a direct connection between the microbiome and associated plant metabolome. In this way, the CRISPR-mediated gene-editing tool provides an attractive approach to accomplish the creation of microbial consortia. On the other hand, the genetic manipulation of the host plant with the help of CRISPR-Cas9 can facilitate the characterization and identification of the genetic determinants. It leads to the enhancement of microbial capacity for more trait improvement. Many plant characteristics like phytovolatilization, phytoextraction, phytodesalination and phytodegradation are targeted by these approaches. Alternatively, chemical communications by PGPB are accomplished by the exchange of different signal molecules. For example, quorum-sensing is the way of the cell to cell communication in bacteria that lead to the detection of metabolites produced by pathogens during adverse conditions and also helpful in devising some tactics towards understanding plant immunity. Along with quorum-sensing, different volatile organic compounds and N-acyl homoserine lactones play a significant role in cell to cell communication by microbe to plant and among the plants respectively. Therefore, it is necessary to get details of all the significant approaches that are useful in exploring cell to cell communications. In this review, we have described gene-editing tools and the cell to cell communication process by quorum-sensing based signaling. These signaling processes via CRISPR- Cas9 mediated gene editing can improve the microbe-plant community in adverse climatic conditions.     

Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.     

Analysis of Human Blood Plasma Proteome from Ten Healthy Volunteers from Indian Population

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used ‘reference plasma sample’, a pool of plasma from 10 healthy individuals from Indian population in the age group of 25–60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by≥2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.