A N Bach Institute of Biochemistry

New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

The mechanism of operation of laccase-mediator systems (LMSs) in xenobiotic degradation mediated by "true" redox mediators and laccase enhancing agents is considered. Structural formulae of most common laccase mediators and compounds that can be used as agents enhancing the enzyme operation are presented. Examples of LMS application in biotechnology are described.

For d-dimensional tensors with possibly large $d>3$, an hierarchical data structure, called the Tree-Tucker format, is presented as an alternative to the canonical decomposition. It has asymptotically the same (and often even smaller) number of representation parameters and viable stability properties. The approach involves a recursive construction described by a tree with the leafs corresponding to the Tucker decompositions of three-dimensional tensors, and is based on a sequence of SVDs for the recursively obtained unfolding matrices and on the auxiliary dimensions added to the initial “spatial” dimensions. It is shown how this format can be applied to the problem of multidimensional convolution. Convincing numerical examples are given.

This review concerns copper-containing oxidases--laccases. Principal biochemical and electrochemical properties of laccases isolated from different sources are described, as well as their structure and mechanism of catalysis. Possible applications of laccases in different fields of biotechnology are discussed.

"All stood amazed, until an old woman, tottering out from among the crowd, put her hand to her brow, and peering under it in his face for a moment exclaimed, 'Sure enough! it is Rip Van Winkle-- it is himself. Welcome home again, old neighbour-- Why, where have you been these twenty long years?"'

The Nelson-Somogyi (NS) and 3,5-dinitrosalicylic acid (DNS) assays for reducing sugars are widely used in measurements of carbohydrase activities against different polysaccharides. Using twelve commercial enzyme preparations, the comparison of the NS and DNS assays in determination of cellulase, β-glucanase, xylanase, and β-mannanase activities was carried out. When cellulase activities against CMC were measured, the DNS assay gave activity values, which were typically 40-50% higher than those obtained with the NS assay. In the analysis of the xylanase, β-mannanase, and β-glucanase activities, the overestimations by the DNS assay were much more pronounced (the observed differences in the activities were 3- to 13-fold). Reasons for preferential use of the NS assay for measuring activities of carbohydrases other than cellulases are discussed.

Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.

Research Article| August 01 1994 NAD+-dependent formate dehydrogenase V O Popov; V O Popov 1A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskiy pr. 33, 117071, Moscow, Russia Search for other works by this author on: This Site PubMed Google Scholar V S Lamzin V S Lamzin 1A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskiy pr. 33, 117071, Moscow, Russia Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Online ISSN: 1470-8728 Print ISSN: 0264-6021 © 1994 The Biochemical Society, London1994 Biochem J (1994) 301 (3): 625–643. https://doi.org/10.1042/bj3010625 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation V O Popov, V S Lamzin; NAD+-dependent formate dehydrogenase. Biochem J 1 August 1994; 301 (3): 625–643. doi: https://doi.org/10.1042/bj3010625 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1994 The Biochemical Society, London1994 Article PDF first page preview Close Modal You do not currently have access to this content.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.

The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.

Reactive oxygen species (ROS) are formed by fungi in the course of metabolic activity. ROS production increases in fungi due to various stress agents such as starvation, light, mechanical damage, and interactions with some other living organisms. Regulation of ROS level appears to be very important during development of the fungal organism. ROS sources in fungal cells, their sensors, and ROS signal transduction pathways are discussed in this review. Antioxidant defense systems in different classes of fungi are characterized in detail. Particular emphasis is placed on ROS functions in interactions of phytopathogenic fungi with plant cells.

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTTrends in biochemistry and enzymology of cellulose degradationA. A. KlyosovCite this: Biochemistry 1990, 29, 47, 10577–10585Publication Date (Print):November 1, 1990Publication History Published online1 May 2002Published inissue 1 November 1990https://doi.org/10.1021/bi00499a001Request reuse permissionsArticle Views1219Altmetric-Citations144LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alertsclose Get e-Alerts

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O2 flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.

After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of "non-culturable" cells of the "Academia" strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton's medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4-5 months post-inoculation, of a homogeneous population of ostensibly "non-culturable" cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10(5) organisms ml(-1), and this value was further increased by one log using supernatant from an actively growing culture. Populations of "non-culturable" cells of Mycobacterium tuberculosis were also obtained by the filtration of "clumpy" cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The "non-culturable" cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with "non-culturable" bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.

We consider Tucker-like approximations with an $r \times r \times r$ core tensor for three-dimensional $n \times n \times n$ arrays in the case of $r \ll n$ and possibly very large n (up to $10^4$–$10^6$). As the approximation contains only $\mathcal{O}(rn + r^3)$ parameters, it is natural to ask if it can be computed using only a small amount of entries of the given array. A similar question for matrices (two-dimensional tensors) was asked and positively answered in [S. A. Goreinov, E. E. Tyrtyshnikov, and N. L. Zamarashkin, A theory of pseudo-skeleton approximations, Linear Algebra Appl., 261 (1997), pp. 1–21]. In the present paper we extend the positive answer to the case of three-dimensional tensors. More specifically, it is shown that if the tensor admits a good Tucker approximation for some (small) rank r, then this approximation can be computed using only $\mathcal{O}(nr)$ entries with $\mathcal{O}(nr^{3})$ complexity.

Point-of-care (POC) or bedside analysis is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufacturing technology for lateral flow (immunochromatographic) test strips. They are widely used to rapidly and easily control a variety of biomarkers of infectious diseases and metabolic and functional disorders, as well as in consumer protection and environmental monitoring. However, traditional lateral flow tests rely on visual assessment and qualitative conclusion, which limit the objectivity and information output of the assays. Therefore, there is a need for approaches that retain the advantages of lateral flow assays and provide reliable quantitative information about the content of a target compound in a sample mixture. This review describes the main options for detecting, processing, and interpreting immunochromatographic analysis results. The possibilities of modern portable detectors that register colored, fluorescent, magnetic, and conductive labels are discussed. Prospects for further development in this direction are also examined.

Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the 'Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.

Benzothiazinones (BTZs) are antituberculosis drug candidates with nanomolar bactericidal activity against tubercle bacilli. Here we demonstrate that BTZs are suicide substrates of the FAD-dependent decaprenylphosphoryl-β-D-ribofuranose 2'-oxidase DprE1, an enzyme involved in cell-wall biogenesis. BTZs are reduced by DprE1 to an electrophile, which then reacts in a near-quantitative manner with an active-site cysteine of DprE1, thus providing a rationale for the extraordinary potency of BTZs. Mutant DprE1 enzymes from BTZ-resistant strains reduce BTZs to inert metabolites while avoiding covalent inactivation. Our results explain the basis for drug sensitivity and resistance to an exceptionally potent class of antituberculosis agents.