Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both: AN EXAMPLE OF MOLECULAR SYMBIOSIS*

Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex formed by two pathway enzymes: chorismate mutase (CM) and 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Whereas both enzymes are active in isolation, the catalytic activity of both enzymes is enhanced, and in particular that of the much smaller CM is greatly enhanced (by 120-fold), by formation of a hetero-octameric complex between CM and DAH7PS. Moreover, on complex formation M. tuberculosis CM, which has no allosteric response on its own, acquires allosteric behavior to facilitate its own regulatory needs by directly appropriating and partly reconfiguring the allosteric machinery that provides a synergistic allosteric response in DAH7PS. Kinetic and analytical ultracentrifugation experiments demonstrate that allosteric binding of phenylalanine specifically promotes hetero-octameric complex dissociation, with concomitant reduction of CM activity. Together, DAH7PS and CM from M. tuberculosis provide exquisite control of aromatic amino acid biosynthesis, not only controlling flux into the start of the pathway, but also directing the pathway intermediate chorismate into either Phe/Tyr or Trp biosynthesis.     

Medical ethnobotany of the Albanian Alps in Kosovo

Background

The boreal forest of Canada is home to several hundred thousands Aboriginal people who have been using medicinal plants in traditional health care systems for thousands of years. This knowledge, transmitted by oral tradition from generation to generation, has been eroding in recent decades due to rapid cultural change. Until now, published reviews about traditional uses of medicinal plants in boreal Canada have focused either on particular Aboriginal groups or on restricted regions. Here, we present a review of traditional uses of medicinal plants by the Aboriginal people of the entire Canadian boreal forest in order to provide comprehensive documentation, identify research gaps, and suggest perspectives for future research.

Methods

A review of the literature published in scientific journals, books, theses and reports.

Results

A total of 546 medicinal plant taxa used by the Aboriginal people of the Canadian boreal forest were reported in the reviewed literature. These plants were used to treat 28 disease and disorder categories, with the highest number of species being used for gastro-intestinal disorders, followed by musculoskeletal disorders. Herbs were the primary source of medicinal plants, followed by shrubs. The medicinal knowledge of Aboriginal peoples of the western Canadian boreal forest has been given considerably less attention by researchers. Canada is lacking comprehensive policy on harvesting, conservation and use of medicinal plants. This could be explained by the illusion of an infinite boreal forest, or by the fact that many boreal medicinal plant species are widely distributed.

Conclusion

To our knowledge, this review is the most comprehensive to date to reveal the rich traditional medicinal knowledge of Aboriginal peoples of the Canadian boreal forest. Future ethnobotanical research endeavours should focus on documenting the knowledge held by Aboriginal groups that have so far received less attention, particularly those of the western boreal forest. In addition, several critical issues need to be addressed regarding the legal, ethical and cultural aspects of the conservation of medicinal plant species and the protection of the associated traditional knowledge.     

Polymorphic Variants of SCN1A and EPHX1 Influence Plasma Carbamazepine Concentration,Metabolism and Pharmacoresistance in a Population of Kosovar Albanian Epileptic Patients

Aim

The present study aimed to evaluate the effects of gene variants in key genes influencing pharmacokinetic and pharmacodynamic of carbamazepine (CBZ) on the response in patients with epilepsy.

Materials & Methods

Five SNPs in two candidate genes influencing CBZ transport and metabolism, namely ABCB1 or EPHX1, and CBZ response SCN1A (sodium channel) were genotyped in 145 epileptic patients treated with CBZ as monotherapy and 100 age and sex matched healthy controls. Plasma concentrations of CBZ, carbamazepine-10,11-epoxide (CBZE) and carbamazepine-10,11-trans dihydrodiol (CBZD) were determined by HPLC-UV-DAD and adjusted for CBZ dosage/kg of body weight.

Results

The presence of the SCN1A IVS5-91G>A variant allele is associated with increased epilepsy susceptibility. Furthermore, carriers of the SCN1A IVS5-91G>A variant or of EPHX1 c.337T>C variant presented significantly lower levels of plasma CBZ compared to carriers of the common alleles (0.71±0.28 vs 1.11±0.69 μg/mL per mg/Kg for SCN1A IVS5-91 AA vs GG and 0.76±0.16 vs 0.94±0.49 μg/mL per mg/Kg for EPHX1 c.337 CC vs TT; P<0.05 for both). Carriers of the EPHX1 c.416A>G showed a reduced microsomal epoxide hydrolase activity as reflected by a significantly decreased ratio of CBZD to CBZ (0.13±0.08 to 0.26±0.17, p<0.05) also of CBZD to CBZE (1.74±1.06 to 3.08±2.90; P<0.05) and CDR_CBZD (0.13±0.08 vs 0.24±0.19 μg/mL per mg/Kg; P<0.05). ABCB1 3455C>T SNP and SCN1A 3148A>G variants were not associated with significant changes in CBZ pharmacokinetic. Patients resistant to CBZ treatment showed increased dosage of CBZ (657±285 vs 489±231 mg/day; P<0.001) but also increased plasma levels of CBZ (9.84±4.37 vs 7.41±3.43 μg/mL; P<0.001) compared to patients responsive to CBZ treatment. CBZ resistance was not related to any of the SNPs investigated.

Conclusions

The SCN1A IVS5-91G>A SNP is associated with susceptibility to epilepsy. SNPs in EPHX1 gene are influencing CBZ metabolism and disposition. CBZ plasma levels are not an indicator of resistance to the therapy.     

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P?<?0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20–50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.     

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation,Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus

Origins of Life and Evolution of Biospheres - Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum,...     

Irreversible inactivation of human erythrocyte pyruvate kinase by 2,3-butanedione

Human erythrocyte pyruvate kinase was found to be irreversibly inactivated by butanedione in the dark. The second-order rate constants for inactivation at pH 8.0 and 25 degrees C were 2.14 and 2.74 M-1 min-1 in the absence and presence of 50 mM borate, respectively. The pH profile of the inactivation indicated the involvement of a residue with an apparent pK alpha of 8.1-8.3. ADP and phosphoenolpyruvate acted as partial inhibitors of the inactivation process. Certain details of the inactivation, spectral studies, and fluorometric determinations gave evidence for arginine as the only target residue. A total of 23 +/- 3 residues per subunit were modified within the period required for inactivation. In the same period the presence of 4 mM ADP reduced the extent of inactivation by 70% and the number of modified residues to 18 +/- 4. The number of the arginine residues protected by ADP from butanedione modification was 5.0 +/- 1.3 per subunit.     

Structural and biophysical characterization of an antimicrobial peptide chimera comprised of lactoferricin and lactoferrampin

Lactoferricin and lactoferrampin are two antimicrobial peptides found in the N-terminal lobe of bovine lactoferrin with broad spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacteria as well as Candida albicans. A heterodimer comprised of lactoferrampin joined to a fragment of lactoferricin was recently reported in which these two peptides were joined at their C-termini through the two amino groups of a single Lys residue (Bolscher et al., 2009, Biochimie 91(1):123-132). This hybrid peptide, termed LFchimera, has significantly higher antimicrobial activity compared to the individual peptides or an equimolar mixture of the two. In this work, the underlying mechanism behind the increased antibacterial activity of LFchimera was investigated. Differential scanning calorimetry studies demonstrated that all the peptides influenced the thermotropic phase behaviour of anionic phospholipid suspensions. Calcein leakage and vesicle fusion experiments with anionic liposomes revealed that LFchimera had enhanced membrane perturbing properties compared to the individual peptides. Peptide structures were evaluated using circular dichroism and NMR spectroscopy to gain insight into the structural features of LFchimera that contribute to the increased antimicrobial activity. The NMR solution structure, determined in a miscible co-solvent mixture of chloroform, methanol and water, revealed that the Lys linkage increased the helical content in LFchimera compared to the individual peptides, but it did not fix the relative orientations of lactoferricin and lactoferrampin with respect to each other. The structure of LFchimera provides insight into the conformation of this peptide in a membranous environment and improves our understanding of its antimicrobial mechanism of action.     

Ultrastructural effects of antimicrobial peptides from bovine lactoferrin on the membranes of Candida albicans and Escherichia coli

Antimicrobial peptides allegedly exert their action on microbial membranes. Bovine lactoferrin enfold two antimicrobial domains, lactoferricin B (LFcin B) and lactoferrampin (LFampin). Effects of representative peptides thereof on the membranes of Candida albicans and Escherichia coli were investigated. Confocal laser scanning microscopy revealed that these peptides were internalized within a few minutes, concurrently with disrupting membrane integrity as indicated by freeze-fracture transmission electron microscopy. The most striking findings were induction of distinct vesicle-like structures in the membrane of C. albicans by the LFampin peptide, and detachment of the outer membrane and surface protrusions in E. coli by the LFcin B peptide.     

S100P is a novel interaction partner and regulator of IQGAP1

Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 μM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.