Comparison of effects of naloxone and catecholamines on acid-base balance in canine hemorrhagic shock

The present study was conducted to compare the effect of naloxone, an opiate receptor antagonist, with catecholamines on acid-base status and survival in dogs subjected to hemorrhagic shock. Arterial lactic acid concentration which had increased during hemorrhage, decreased significantly (P less than 0.05) in naloxone treated animals but increased further in catecholamine treated dogs. Blood bicarbonate concentration and PCO2 which had markedly decreased 1 hr after hemorrhage recovered significantly (P less than 0.05) in naloxone group of animals. On the other hand bicarbonate and pH declined further in noradrenaline group and remained unchanged in dopamine group. These results as well as better survival rate observed in naloxone treated animals suggest the superiority of naloxone over dopamine and noradrenaline, as an adjunct to blood transfusion in the treatment of hemorrhagic shock.     

Distinct mechanisms underlie pattern formation in the skin and skin appendages

Patterns form with the break of homogeneity and lead to the emergence of new structure or arrangement. There are different physiological and pathological mechanisms that lead to the formation of patterns. Here, we first introduce the basics of pattern formation and their possible biological basis. We then discuss different categories of skin patterns and their potential underlying molecular mechanisms. Some patterns, such as the lines of Blaschko and Naevus, are based on cell lineage and genetic mosaicism. Other patterns, such as regionally specific skin appendages, can be set by distinct combinatorial molecular codes, which in turn may be set by morphogenetic gradients. There are also some patterns, such as the arrangement of hair follicles (hair whorls) and fingerprints, which involve genetics as well as stochastic epigenetic events based on physiochemical principles. Many appendage primordia are laid out in developmental waves. In the adult, some patterns, such as those involving cycling hair follicles, may appear as traveling waves in mice. Since skin appendages can renew themselves in regeneration, their size and shape can still change in the adult via regulation by hormones and the environment. Some lesion patterns are based on pathological changes involving the above processes and can be used as diagnostic criteria in medicine. Understanding the different mechanisms that lead to patterns in the skin will help us appreciate their full significance in morphogenesis and medical research. Much remains to be learned about complex pattern formation, if we are to bridge the gap between molecular biology and organism phenotypes.     

Mathematical modelling of tumour acidity

Acid-mediated tumour invasion is receiving increasing experimental and clinical attention. Previous models proposed to describe this phenomenon failed to capture key properties of the system, such as the existence of the benign steady state, or predicted incorrectly the size of the inter-tissue gap. Here we show that taking proper account of quiescence ameliorates these drawbacks as well as revealing novel behaviour. The simplicity of the model allows us to fully identify the key parameters controlling different aspects of behaviour.     

Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center

Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG codons at predetermined positions + β-alanyl–tRNA_CUA, the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted β-amino acids, and direct evidence for β-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted β-amino acids, including α-methyl-β-alanine, β,β-dimethyl-β-alanine, β-phenylalanine, and β-(p-bromophenyl)alanine. The β-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of β-alanine and representative α-l-amino acids. The isolated proteins containing the modified β-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the β-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of β,β-dimethyl-β-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the β-amino acid.     

Comparing a discrete and continuum model of the intestinal crypt

The integration of processes at different scales is a key problem in the modelling of cell populations. Owing to increased computational resources and the accumulation of data at the cellular and subcellular scales, the use of discrete, cell-level models, which are typically solved using numerical simulations, has become prominent. One of the merits of this approach is that important biological factors, such as cell heterogeneity and noise, can be easily incorporated. However, it can be difficult to efficiently draw generalizations from the simulation results, as, often, many simulation runs are required to investigate model behaviour in typically large parameter spaces. In some cases, discrete cell-level models can be coarse-grained, yielding continuum models whose analysis can lead to the development of insight into the underlying simulations. In this paper we apply such an approach to the case of a discrete model of cell dynamics in the intestinal crypt. An analysis of the resulting continuum model demonstrates that there is a limited region of parameter space within which steady-state (and hence biologically realistic) solutions exist. Continuum model predictions show good agreement with corresponding results from the underlying simulations and experimental data taken from murine intestinal crypts.     

Ab initio identification of novel regulatory elements in the genome of Trypanosoma brucei by Bayesian inference on sequence segmentation

Background

The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes.

Methodology/Principle Findings

Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site.

Conclusions/Significance

The nature of the elements discovered reinforces the hypothesis that context dependant RNA secondary structure has an important influence on gene expression regulation in Trypanosoma brucei.     

The Single Substitution I259T,Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches,Enhances Fibrinolytic Activity

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla β-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the β-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in α₂-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution ²⁵⁹IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution ²⁵⁹TIDKN/IIDKT in Pla altered proteolysis in favor of α₂-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.Since the year 540, plague has killed some 200 million humans in three pandemics, i.e., the Justinian plague, the Black Death, and the modern plague (36). Genomic studies have estimated that the etiological agent, Yersinia pestis, evolved from the oral-fecal pathogen Yersinia pseudotuberculosis serotype O1b only shortly before the first pandemic, i.e., 5,000 to 20,000 years ago (1, 2, 46), which has made the bacterium a paradigm of the rapid evolution of a severe bacterial pathogen (57). At least four biovars of Y. pestis have been identified through metabolic and genomic studies; of these biovars, Antiqua, Medievalis, and Orientalis may be associated with the three plague pandemics, whereas the fourth biovar, Microtus, is associated with human-attenuated Y. pestis strains from two geographically distant infection foci in China (36, 59-61). A recent molecular analysis indicated that the biovars are not monophyletic and proposed the subdivision of Y. pestis into eight molecular groupings, which represent different evolutionary branches and histories and are only partially compatible with the biovars (1). Y. pestis evolved from Y. pseudotuberculosis along branch 0, which consists of “atypical” Y. pestis strains designated Angola, Microtus, and Pestoides; these are phylogenetically ancestral to the Antiqua, Medievalis, and Orientalis branches (1).As a disease, plague exhibits various pathologies. Bubonic plague is the zoonotic form of the disease, which is usually acquired by humans from the bite of a flea that has been infected through a blood meal on a diseased rodent (36). The bacteria invade at the intradermal flea bite site and migrate to lymphatic vessels and then to regional draining lymph nodes, where they multiply and cause the development of buboes (44). Without early treatment, bubonic plague progresses to life-threatening septicemic plague, and hematogenous spread of the bacterium to lungs leads to pneumonic plague, a rapidly fatal and highly contagious airborne disease. Occasional injection of Y. pestis cells by the flea directly into the circulatory system leads to primary septicemic plague (43).The plasminogen activator Pla is a cell surface protease encoded by the Y. pestis-specific plasmid pPCP1 (10, 48). Pla is essential in the pathogenesis of bubonic (43, 49) and pneumonic plague (28), whereas it has less of a role in primary septicemic plague (43, 49). The pla gene is highly transcribed in buboes of Y. pestis-infected mice (45), and Pla specifically potentiates migration of the bacteria to lymphatic tissue (43). Pla seems to have a different role in pneumonic plague, where it allows Y. pestis to replicate rapidly in the lungs, causing lethal fulminant pneumonia (28). Virulent Y. pestis strains lacking the Pla-encoding plasmid pPCP1 have been isolated in Asia (3), and they can be associated with primary septicemic plague (43).Pla is an aspartic protease (22, 55) that activates human plasminogen (Plg) to the serine protease plasmin (47) and inactivates the plasmin inhibitor α₂-antiplasmin (α₂AP), thus affecting the main control system for plasmin activity (22). Plg is an abundant circulating zymogen, and its activation is central in the pathogenesis of plague (13, 28, 43), and plasmin is a powerful serine protease associated with cell migration and degradation of fibrin clots (29, 32, 37). In accordance with this, Pla-mediated bacterial adherence directs uncontrolled plasmin proteolysis onto basement membranes to enhance bacterial metastasis through tissue barriers (25, 27), and fibrinolysis by Pla-generated plasmin activity plays a role in the pathogenesis of bubonic plague (8).Compared to those of other Y. pestis biovars, Microtus isolates have several unique genomic features that may be involved in their inherent inability to attack the human host, and specific losses of genes or gene functions are thought to be responsible for the human attenuation (59). Interestingly, the attenuation does not apply to the murine host. The predicted amino acid sequence of the Pla polypeptide is remarkably conserved: in the branches Antiqua, Medievalis, and Orientalis, the Pla sequences are completely identical, whereas a single amino acid substitution, T259I, has been detected in atypical Angola and Microtus strains (6, 38, 50). A genetic analysis of 260 isolates of Y. pestis showed that the T259I substitution in Pla is shared by all isolates of biovar Microtus but absent in those of other biovars (59). Many of the Pestoides strains lack the pPCP1 plasmid and hence also the pla gene (12), and pla sequences from Pestoides are not available.Pla is a member of the omptin family of conserved outer membrane proteases/adhesins detected in several gram-negative bacterial pathogens (15, 17, 21). The omptins have the same molecular size, a β-barrel fold of 10 transmembrane β strands, and five surface-exposed loops, L1 to L5 (Fig. ​(Fig.1).1). The catalytic residues and the residues interacting with lipid A in the outer membrane are completely conserved (17, 21-23, 41, 55). The omptins cleave peptide substrates at basic residues (17) but show dramatic heterogeneity in the recognition of biologically important polypeptides, such as Plg, the antiprotease α₂AP, gelatin, and progelatinases. Analyses of hybrid proteins created between Pla and the omptins PgtE of Salmonella enterica and OmpT of Escherichia coli have indicated that the differing polypeptide substrate selectivity of omptins is dictated by sequence variation in the mobile loop structures of the β-barrel (22, 40). Residue T259 in Pla is located at surface loop 5 and oriented inward in the active-site groove of the Pla barrel, close to residue K262, where Pla is autoprocessed (22, 23) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Model of Pla structure (23) and location of residue Thr259. Side (top drawing) and top (bottom drawing) views of the transmembrane β-barrel are shown. L1 to L5 are the surface loops. Catalytic residues Asp84, Asp86, Asp206, and His208 are indicated in green, Thr259 is in red, and the autoprocessing site Lys262 is in yellow. OM is the outer membrane. (C) Amino acid sequence of residues 254 to 273 at L5 and the termini of β-strands 9 and 10 in Pla, Microtus Pla, and PgtE are shown.The omptin β-barrel has spread by horizontal gene transfer in gram-negative bacteria and adapted to the life-styles of host bacteria (15, 17, 21, 22, 40). Overall, the omptins give an example of an evolvable, robust enzyme fold (34) that easily acquires novel or improved functions. The fact that the single substitution T259I associates with ancestral Y. pestis Microtus and Angola populations suggests that Microtus Pla represents a form of the protein that preceded the common Pla protein. The central role of Plg activation in the pathogenesis of plague led us to analyze whether the single substitution T259I affects the fibrinolytic activities of the Pla molecule.     

Angiogenesis and vascular remodelling in normal and cancerous tissues

Vascular development and homeostasis are underpinned by two fundamental features: the generation of new vessels to meet the metabolic demands of under-perfused regions and the elimination of vessels that do not sustain flow. In this paper we develop the first multiscale model of vascular tissue growth that combines blood flow, angiogenesis, vascular remodelling and the subcellular and tissue scale dynamics of multiple cell populations. Simulations show that vessel pruning, due to low wall shear stress, is highly sensitive to the pressure drop across a vascular network, the degree of pruning increasing as the pressure drop increases. In the model, low tissue oxygen levels alter the internal dynamics of normal cells, causing them to release vascular endothelial growth factor (VEGF), which stimulates angiogenic sprouting. Consequently, the level of blood oxygenation regulates the extent of angiogenesis, with higher oxygenation leading to fewer vessels. Simulations show that network remodelling (and de novo network formation) is best achieved via an appropriate balance between pruning and angiogenesis. An important factor is the strength of endothelial tip cell chemotaxis in response to VEGF. When a cluster of tumour cells is introduced into normal tissue, as the tumour grows hypoxic regions form, producing high levels of VEGF that stimulate angiogenesis and cause the vascular density to exceed that for normal tissue. If the original vessel network is sufficiently sparse then the tumour may remain localised near its parent vessel until new vessels bridge the gap to an adjacent vessel. This can lead to metastable periods, during which the tumour burden is approximately constant, followed by periods of rapid growth.