Cloning of DNA sequences encoding foreign peptides and their expression in the K88 pili

A genetic system that allows the cloning of a peptide-coding sequence in the Escherichia coli K88ac and K88ad pilin genes and their expression as recombinant pili has been constructed. Two insertion vectors were created by subcloning the pilin genes in a pBR322 plasmid and replacing the coding sequence of two nonconserved clusters by a linker. The K88ac helper genes were subcloned in the compatible pACYC184 plasmid, and expression of pili by bacteria carrying both plasmids occurred by complementation. Two peptide-coding sequences of the influenza hemagglutinin were cloned in both insertion vectors, and recombinant pilins were shown to be assembled in pili. One recombinant pilus was shown to elicit antibodies against the synthetic peptide in immunized rats. The somatostatin-coding sequence was cloned in both vectors and led in one case to detectable pilus production. The fused somatostatin was shown to be recognized by specific monoclonal and polyclonal antibodies.     

Electrostatically biased binding of kinesin to microtubules

The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.     

Experimental design to optimize an <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid

The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.     

Monosodium glutamate raises antral distension and plasma amino acid after a standard meal in humans

The consumption of monosodium glutamate (MSG) is advocated to elicit physiological and metabolic effects, yet these effects have been poorly investigated directly in humans and in particular in the postprandial phase. Thirteen healthy adults were supplemented for 6 days with a nutritional dose of MSG (2 g) or sodium chloride (NaCl) as control, following a crossover design. On the 7th day, they underwent a complete postprandial examination for the 6 h following the ingestion of the same liquid standard meal (700 kcal, 20% of energy as [(15)N]protein, 50% as carbohydrate, and 30% as fat) supplemented with MSG or NaCl. Real-ultrasound measures of antral area indicated a significant increased distension for the 2 h following the meal supplemented with MSG vs. NaCl. This early postprandial phase was also associated with significantly increased levels of circulating leucine, isoleucine, valine, lysine, cysteine, alanine, tyrosine, and tryptophan after MSG compared with NaCl. No changes to the postprandial glucose, insulin, glucagon-like peptide (GLP)-1, and ghrelin were noted between MSG- and NaCl-supplemented meals. Subjective assessments of hunger and fullness were neither affected by MSG supplementation. Finally, the postprandial fate of dietary N was identical between dietary conditions. Our findings indicate that nutritional dose of MSG promoted greater postprandial elevations of several indispensable amino acids in plasma and induced gastric distension. Further work to elucidate the possible sparing effect of MSG on indispensable amino acid first-pass uptake in humans is warranted. This trial was registered at clinicaltrials.gov as NCT00862017.     

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis?trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.     

Evidence of multifractality from emerging European stock markets

We test for the presence of multifractality in the daily returns of the three most important stock market indices from Central and Eastern Europe, Czech PX, Hungarian BUX and Polish WIG using the Empirical Mode Decomposition based Multifractal Detrended Fluctuation Analysis. We found that the global Hurst coefficient varies with the q coefficient and that there is multifractality evidenced through the multifractal spectrum. The exercise is replicated for the sample around the high volatility period corresponding to the last global financial crisis. Although no direct link has been found between the crisis and the multifractal spectrum, the crisis was found to influence the overall shape as quantified through the norm of the multifractal spectrum.     

Biocomposite films prepared from ionic liquid solutions of chitosan and cellulose

Blends of chitosan and cellulose were successfully produced using 1-butyl-3-methylimidazolium acetate (BMIMAc) as solvent media. Films were prepared from the blends by manually spreading the solution on a flat surface and precipitating the polymers in a mixture of methanol and water. To prevent the shrinkage of films, most of the absorbed water was removed by freeze drying under vacuum. Films prepared from the polymeric solutions were investigated by means of FT-IR, TGA, X-ray diffraction and SEM measurements. The shifting of the bands corresponding to -NH and CO groups of chitosan (FT-IR), the absence of the diffraction peaks at 2θ = 10.7 and 14.9° (XRD), the increased E_a for thermal decomposition for all the polymeric blends (MTGA), and the presence of an apparent homogeneous structure with no phase separation of the two polymers (SEM) provide evidence for the miscibility between chitosan and cellulose in the solid state.