Robust morphogenesis by chaotic dynamics.

This research illustrates that complex dynamics of gene products enable the creation of any prescribed cellular differentiation patterns. These complex dynamics can take the form of chaotic, stochastic, or noisy chaotic dynamics. Based on this outcome and previous research, it is established that a generic open chemical reactor can generate an exceptionally large number of different cellular patterns. The mechanism of pattern generation is robust under perturbations and it is based on a combination of Turing's machines, Turing instability and L. Wolpert's gradients. These results can help us to explain the formidable adaptive capacities of biochemical systems.

Rise of nations: Why do empires expand and fall?

We consider centralized networks composed of multiple satellites arranged around a few dominating super-egoistic centers. These so-called empires are organized using a divide and rule framework enforcing strong center-satellite interactions while keeping the pairwise interactions between the satellites sufficiently weak. We present a stochastic stability analysis, in which we consider these dynamical systems as stable if the centers have sufficient resources while the satellites have no value. Our model is based on a Hopfield type network that proved its significance in the field of artificial intelligence. Using this model, it is shown that the divide and rule framework provides important advantages: it allows for completely controlling the dynamics in a straight-forward way by adjusting center-satellite interactions. Moreover, it is shown that such empires should only have a single ruling center to provide sufficient stability. To survive, empires should have switching mechanisms implementing adequate behavior models by choosing appropriate local attractors in order to correctly respond to internal and external challenges. By an analogy with Bose-Einstein condensation, we show that if the noise correlations are negative for each pair of nodes, then the most stable structure with respect to noise is a globally connected network. For social systems, we show that controllability by their centers is only possible if the centers evolve slowly. Except for short periods when the state approaches a certain stable state, the development of such structures is very slow and negatively correlated with the size of the system's structure. Hence, increasing size eventually ends up in the "control trap."

Punctuated evolution and robustness in morphogenesis.

This paper presents an analytic approach to the pattern stability and evolution problem in morphogenesis. The approach used here is based on the ideas from the gene and neural network theory. We assume that gene networks contain a number of small groups of genes (called hubs) controlling morphogenesis process. Hub genes represent an important element of gene network architecture and their existence is empirically confirmed. We show that hubs can stabilize morphogenetic pattern and accelerate the morphogenesis. The hub activity exhibits an abrupt change depending on the mutation frequency. When the mutation frequency is small, these hubs suppress all mutations and gene product concentrations do not change, thus, the pattern is stable. When the environmental pressure increases and the population needs new genotypes, the genetic drift and other effects increase the mutation frequency. For the frequencies that are larger than a critical amount the hubs turn off; and as a result, many mutations can affect phenotype. This effect can serve as an engine for evolution. We show that this engine is very effective: the evolution acceleration is an exponential function of gene redundancy. Finally, we show that the Eldredge-Gould concept of punctuated evolution results from the network architecture, which provides fast evolution, control of evolvability, and pattern robustness. To describe analytically the effect of exponential acceleration, we use mathematical methods developed recently for hard combinatorial problems, in particular, for so-called k-SAT problem, and numerical simulations.

Critical involvement of a carbamylated lysine in catalytic function of class D beta-lactamases.

beta-Lactamases are the resistance enzymes for beta-lactam antibiotics, of which four classes are known. beta-lactamases hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. It is shown herein that the class D OXA-10 beta-lactamase depends critically on an unusual carbamylated lysine as the basic residue for both the enzyme acylation and deacylation steps of catalysis. The formation of carbamylated lysine is reversible. Evidence is presented that this enzyme is dimeric and carbamylated in living bacteria. High-resolution x-ray structures for the native enzyme were determined at pH values of 6.0, 6.5, 7.5, and 8.5. Two dimers are present per asymmetric unit. One monomer in each dimer was carbamylated at pH 6.0, whereas all four monomers were fully carbamylated at pH 8.5. At the intermediate pH values, one monomer of each dimer was carbamylated, and the other showed a mixture of carbamylated and non-carbamylated lysines. It would appear that, as the pH increased for the sample, additional lysines were "titrated" by carbamylation. A handful of carbamylated lysines are known from protein crystallographic data, all of which have been attributed roles in structural stabilization (mostly as metal ligands) of the proteins. This paper reports a previously unrecognized role for a noncoordinated carbamylate lysine as a basic residue involved in mechanistic reactions of an enzyme, which indicates another means for expansion of the catalytic capabilities of the amino acids in nature beyond the 20 common amino acids in development of biological catalysts.

From genes to sequences to antibiotics: prospects for future developments from microbial genomics.

The entire genome sequences for a number of microbial organisms have become available over the past few years. This knowledge is the beginning point for understanding the fundamental principles of bacterial structure and function. The prospects for gain in knowledge from genomics are discussed in this report.

A light-inactivated antibiotic.

Sodium 7beta-[(R)-2-(N(b)()-o-nitrobenzyloxycarbonyl)hydrazino-3-pheny lpropa namido]cephalosporanate (1) is described as a new type of beta-lactam antibiotic, which undergoes light-induced destruction of its beta-lactam moiety and hence becomes biologically inactive. This type of antibiotic holds the promise of self-destruction over a number of hours of exposure to light, so that it would not allow selection of resistance in the environment.

Insights into class D beta-lactamases are revealed by the crystal structure of the OXA10 enzyme from Pseudomonas aeruginosa.

BACKGROUND: beta-lactam antibiotic therapies are commonly challenged by the hydrolytic activities of beta-lactamases in bacteria. These enzymes have been grouped into four classes: A, B, C, and D. Class B beta-lactamases are zinc dependent, and enzymes of classes A, C, and D are transiently acylated on a serine residue in the course of the turnover chemistry. While class A and C beta-lactamases have been extensively characterized by biochemical and structural methods, class D enzymes remain the least studied despite their increasing importance in the clinic. RESULTS: The crystal structure of the OXA10 class D beta-lactamase has been solved to 1.66 A resolution from a gold derivative and MAD phasing. This structure reveals that beta-lactamases from classes D and A, despite very poor sequence similarity, share a similar overall fold. An additional beta strand in OXA10 mediates the association into dimers characterized by analytical ultracentrifugation. Major differences are found when comparing the molecular details of the active site of this class D enzyme to the corresponding regions in class A and C beta-lactamases. In the native structure of the OXA10 enzyme solved to 1.8 A, Lys-70 is carbamylated. CONCLUSIONS: Several features were revealed by this study: the dimeric structure of the OXA10 beta-lactamase, an extension of the substrate binding site which suggests that class D enzymes may bind other substrates beside beta-lactams, and carbamylation of the active site Lys-70 residue. The CO2-dependent activity of the OXA10 enzyme and the kinetic properties of the natural OXA17 mutant protein suggest possible relationships between carbamylation, inhibition of the enzyme by anions, and biphasic behavior of the enzyme.

[The dependence of blood and tissue proteolytic activity during acute stress from A type of the organism reaction]. / Zalezhnist' proteolitychnoï aktyvnosti krovi i tkanyn pry hostromu stresi vid typu reahuvannia tvaryn.

In the experiments on rats there was grounded the dependence of proteolytic processes in tissues during acute emotional painful stress from typological peculiarities of the organism that was defined by the method of "Open field".

Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) beta-lactamase from Escherichia coli.

We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased. Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase. Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.

[Effect of contrical on proteolytic enzymes of blood serum in acute emotional-pain stress]. / Vliianie kontrikala na aktivnost' proteoliticheskikh fermentov syvorotki krovi pri ostrom émotsional'no-bolevom stresse.

General proteolytic and antitryptic blood serum activity in acute emotionally-painful stress was investigated as well as that on the background of the before injected contrical. It was determined that in case of a stress there increases the general proteolytic activity of blood serum and at the same time antiproteolytic activity decreases. The coefficient of the relation of antiproteolytic activity to proteolytic activity of blood serum in case of a stress may become of 50% of the control. The introduction of inhibitor of proteinase contrical before the beginning of stressor influence promotes the decrease of general proteolytic activity to the level of the control indexes.

Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene.

Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics. This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.

Isolation and characterization of a species-specific DNA probe for the detection of Candida krusei.

In an attempt to design species-specific primers for the detection of Candida krusei by polymerase chain reaction, a partial genomic DNA library from Candida krusei was screened for hybridization with radiolabeled genomic probes from a broad variety of fungal and bacterial species and from human. Species-specific candidate DNA inserts were then tested for hybridization with dot blots of DNA from various organisms. One 570-basepair insert from Candida krusei DNA that hybridized under stringent conditions only with DNA from Candida krusei and human was sequenced. It revealed considerable homology with the gene for the mitochondrial inner membrane protease I of Saccharomyces cerevisiae, and the 147 amino acid residues deduced from an open reading frame showed considerable homology with the N-terminal portion of the enzyme from Saccharomyces cerevisiae. From the sequence of the Candida krusei DNA fragment, a pair of 21-base oligonucleotide primers enclosing a 501-basepair sequence was designed for polymerase chain reaction. When these primers were tested with a broad range of genomic DNAs, the expected amplification was obtained only with Candida krusei DNA and not with DNA from any other source, including human. Experiments with DNA from mixed cultures of Candida krusei and other yeasts and bacteria showed that the polymerase chain reaction was specific for Candida krusei and that as few as ten cells could be detected.

Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients.

To examine the mechanism of resistance to fluoroquinolones in Mycobacterium tuberculosis, we selected spontaneous fluoroquinolone-resistant mutants from a susceptible strain, H37Rv, and studied the susceptibilities of these mutants and two fluoroquinolone-resistant clinical isolates (A-382, A-564) to various fluoroquinolones and to isoniazid and rifampin. Furthermore, since mutations within the quinolone resistance-determining region of the structural gene encoding the A subunit of DNA gyrase are the most common mechanism of acquired resistance, we amplified this region by PCR and compared the nucleotide sequences of the fluoroquinolone-resistant strains with that of the susceptible strain. Fluoroquinolone-resistant mutants of H37Rv appeared at frequencies of 2 x 10(-6) to 1 x 10(-8). For three mutants selected on ciprofloxacin, ofloxacin, and sparfloxacin, respectively, and the two clinical isolates, MICs of ciprofloxacin and ofloxacin were as high as 16 micrograms/ml, and those of sparfloxacin were 4 to 8 micrograms/ml. They displayed cross-resistance to all fluoroquinolones tested but not to isoniazid or rifampin. Sparfloxacin and FQ-A (PD 127391-0002) were the most potent fluoroquinolones. All of the fluoroquinolone-resistant strains (MICs, > or = 4 micrograms/ml) had mutations in the quinolone resistance-determining region which led to substitution of the Asp residue at position 87 (Asp-87) by Asn or Ala or the substitution of Ala-83 by Val in the A subunit of DNA gyrase. Similar mutations have been noted in other bacterial species and recently in mycobacteria. The broad resistance to fluoroquinolones that arose readily by point mutation in the laboratory and apparently during inadequate therapy, as was the case in the clinical isolates, may ultimately lead to to serious restriction of the use of these drugs in the treatment of tuberculosis.

Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams.

To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 [numbering of Ambler et al. (Biochem J. 267:269-272, 1991)]) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme. All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime. In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam. One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime.

[Cloning and nucleotide sequence determination of the aadB gene from a Salmonella oranienburg strain]. / Klonirovanie i opredelenie nukleotidnoi posledovatel'nosti gena aadB iz shtamma Salmonella oranienburg.

Cloning and nucleotide sequence determination of the aadB gene and boundary DNA fragments from a high molecular weight plasmid of Salmonella oranienburg were performed. The data on the restriction mapping showed that the aadB gene was located within the integrone. Analysis of the nucleotide sequence of the cloned fragment revealed a high level conservative nature of the aadB gene and boundary DNA areas.

[Genes of resistance to aminoglycoside antibiotics in clinical strains of Serratia marcescens and the enzyme encoded by them]. / Geny rezistentnosti k aminoglikozidnym antibiotikam klinicheskikh shtammov Serratia marcescens i kodiruemye imi fermenty.

The patterns of aminoglycoside inactivating enzymes were determined by AGRP in 31 clinical isolated of Serratia marcescens. The results were compared with the data on identification of the aminoglycoside resistance genes by the specific DNA probes. It was shown that all the isolates of Serratia marcescens contained the AAC(6')-Ic gene which was not expressed in some isolates. The other detected aminoglycoside inactivating enzymes were the following: AAC(3)-V in 17 isolates, ANT(2'') in 7 isolates, AAC(3)-I in 4 isolates and APH(3')-I in 13 isolates. Reliability of the methods of AGRP and DNA-DNA hybridization was estimated in the assay of the aminoglycoside resistant clinical strains of Serratia marcescens.