A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope.

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.     

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.     

A para-nitrophenol phosphonate probe labels distinct serine hydrolases of Arabidopsis

Activity-based protein profiling represents a powerful methodology to probe the activity state of enzymes under various physiological conditions. Here we present the development of a para-nitrophenol phosphonate activity-based probe with structural similarities to the potent agrochemical paraoxon. We demonstrate that this probes labels distinct serine hydrolases with the carboxylesterase CXE12 as the predominant target in Arabidopsis thaliana. The designed probe features a distinct labeling pattern and therefore represents a promising chemical tool to investigate physiological roles of selected serine hydrolases such as CXE12 in plant biology.     

Computational Investigations of the Chalcogen-Substituted Carboxylic Acids RC(=O)XH and RC(=X)OH and their Dimers [RC(=O)XH]2 and [RC(=X)OH]2 (X=S, Se, Te)

Semiempirical and ab initio theoretical methods have been used to investigate molecular structures of the chalcogen-substituted carboxylic acid isomers RC(=O)XH (chalcogenol acid) and RC(=X)OH (chalcogenon acid). A recent experimental report suggests that the chalcogenon isomers, although less stable at room temperature, predominate at low temperature in polar solvents and that there is only a small barrier to isomerization between the isomers. Theoretical calculations have been used to locate minimum energy structures of chalcogen-substituted carboxylic acid isomers and to calculate energy differences between pairs of isomers. Carboxylic acids are well known to dimerize, especially in the gas phase and in non-polar solvents. We have, therefore, also calculated energies of dimerization of the chalcogen-substituted acids by optimizing the geometries of the symmetric dimers. We note that the PM3 level of theory is only qualitatively correct for sulfur- and selenium-containing species but fails even qualitatively for the tellurium-containing compounds. Ab initio results confirm the experimental observations and provide good estimates of both isomerization and dimerization energies. We conclude that for many functional groups with tautomers RC(=X)YH and RC(=Y)XH, the more acidic tautomer is the one with the acid proton on the smaller, more electronegative atom, although in many cases this may not be the more stable tautomer.Electronic Supplementary Material available.     

Seasonal changes in the ruminal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus).

The dominant rumen bacteria in high-arctic Svalbard reindeer were characterized, their population densities were estimated, and ruminal pH was determined in summer, when food quality and availability are good, and in winter, when they are poor. In summer the total cultured viable population density was (2.09 +/- 1.26) X 10(10) cells ml-1, whereas in winter it was (0.36 +/- 0.29) X 10(10) cells ml-1, representing a decrease to 17% of the summer population density. On culture, Butyrivibrio fibrisolvens represented 22% of the bacterial population in summer and 30% in winter. Streptococcus bovis represented 17% of the bacterial population in summer but only 4% in winter. Methanogenic bacteria were present at 10(4) cells ml-1 in summer and 10(7) cells ml-1 in winter. In summer and winter, respectively, the proportions of the viable population showing the following activities were as follows: starch utilization, 68 and 63%; fiber digestion, 31 and 74%; cellulolysis, 15 and 35%; xylanolysis, 30 and 58%; proteolysis, 51 and 28%; ureolysis, 40 and 54%; and lactate utilization, 13 and 4%. The principal cellulolytic bacterium was B. fibrisolvens, which represented 66 and 52% of the cellulolytic population in summer and winter, respectively. The results indicate that the microflora of the rumen of Svalbard reindeer is highly effective in fiber digestion and nitrogen metabolism, allowing the animals to survive under the austere nutritional conditions typical of their high-arctic habitat.     

Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Changes in the migration and adhesive activity of polymorphonuclear leukocytes as 1 of the characteristics of Gram-negative bacteria isolated from patients with suppurative lung destruction

The influence of Gram-negative bacteria on the migratory and adhesive activity of polymorphonuclear leukocytes (PMNL) in the peripheral blood of clinically normal donors has been studied by the specially developed method with the use of Boyden chambers. Pseudomonas and enterobacteria have been found to produce complex and various effects on the above-mentioned properties of PMNL. When incubated in fresh serum, Gram-negative bacteria are capable of enhancing the migratory activity of PMNL, this property being least pronounced in P. aeruginosa. The incubation of live bacteria from the authors' collection in the patients' sera or in sera obtained from normal donors and inactivated by heating induces no hemotaxis of PMNL, and P. aeruginosa strains even suppress it under such conditions. The isolated Gram-negative bacteria under study increase the number of highly adhesive PMNL in the population used in this investigation, but P. aeruginosa cultures do not produce such effect.     

Frequency of Erwinia carotovora in the Alyth Burn in eastern Scotland and the sources of the bacterium

Contamination of the Alyth Burn by Erwinia carotovora was monitored monthly over 2 years at nine sites spread over a distance of ca 20 km. The bacterium was detected only once in the upper reaches of the river where it flows in uninhabited moorland but frequency of detection and contamination level tended to increase progressively as the river flowed through the middle reaches mostly in grassland to the lower reaches in arable land where the bacterium was almost always present. Erwinia populations rose from < 10² cells/1 before May to frequently > 10³ but < 10⁴ cells/l thereafter at sites in the arable land zone. A similar pattern was found in the grassland zone except that erwinia numbers were lower. Erwinia numbers at one site in the arable land zone were positively and negatively correlated with the river water temperature and flow rate respectively when there was a 1 month lag between the environmental data and the population recorded. More than 80% of isolates tested were E. carotovora subsp. carotovora.
Water from field drains in arable fields, especially those recently planted with potatoes, was frequently contaminated by E.c. carotovora , with numbers and a temporal pattern similar to those of the Alyth Burn. Drainage water from non-arable fields was rarely contaminated. Infected and rotting potatoes deposited in rivers temporarily contaminated the water. Survival of E. carotovora in dilute phosphate buffer was greater at pH 5˙7 than at pH 7˙7 and they survived for at least 10 d in river water.