Mechanisms of cellulases and xylanases: a detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi

The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates. This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism. Values of kcat are found to be invariant with pH whereas kcat/Km is dependent upon two ionizations of pKa = 4.1 and 7.7. The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types. A strong dependence of both kcat (beta 1g = -1) and kcat/Km (beta 1g = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting. For the cellobiosides a biphasic, concave downward plot is seenj for kcat, indicating a change in rate-determining step across the series. Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k2 and log (k2/Kd) for the cellobiosides of modest (beta 1g = -0.3) slope. These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states. Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta 1g values, reveal that the presence of the distal sugar moiety in cellobiosides results in a less highly charged transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening of supports for the immobilization of pectinmethylesterase from acerola (Malpighia glabra L)

A partially purified extract of pectinmethylesterase (PME) from acerola fruit was immobilized on various supports: glass, celite, chrysotile, agarose, concanavalin A Sepharose 4B, egg shell, polyacrylamide and gelatin. In addition, reticulation with glutaraldehyde was assessed, as well as the use of gelatin in the presence of celite, glass and silica. The highest immobilization yields were obtained when the pectinmethylesterase was immobilized in concanavalin A Sepharose 4B (81.7%) and in gelatin‐water (78.0%). Copyright © 2004 Society of Chemical Industry     

Electrical Conductivity Measurement Through the Loaded Q Factor of a Resonant Cavity

We present a method for measuring the electrical conductivity of metallic materials that relies on the ratio of two loaded Q factors, Q_R/Q_X, with Q_R corresponding to a TE₀₁₁-mode reference cavity made of aluminum, and Q_X the Q that results upon replacing the aluminum plate with the one fabricated from the material to be examined. Electrical conductivity is mathematically inferred from the ratio Q_R/Q_X where the loaded Q factors are measured by using the transmission-type method. Within a 3.0 percent accuracy, conductivities determined at 8.7 GHz for electrolytic copper (5.6 times 10⁷ S/m) and brass (1.6 times 10⁷ S/m) show to be in good agreement with those reported in the literature.     

Liquid–liquid extraction of a recombinant protein with reverse micelles

Recombinant cytochrome b₅ was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase. The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein. This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis. The back-extraction of cytochrome b₅ to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1–2 mol dm^?3 NaCl) and low temperatures (4°C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.     

Comparison of the genomic short regions of a vaccine strain (SA-2) and a virulent strain (CSW-1) of infectious laryngotracheitis virus (Gallid herpesvirus 1)

Restriction enzyme linkage maps were produced for the genomic short region of the virulent infectious laryngotracheitis virus (CSW-1 strain). After comparison with the equivalent restriction enzyme linkage maps for the infectious laryngotracheitis virus SA-2 strain (a vaccine strain), it was determined that the maps for the short regions of the two strains were identical, apart from a single section in each of the inverted terminal repeats. Each inverted terminal repeat of the SA-2 strain was discovered to contain 467 base pairs more DNA than the CSW-1 strain's inverted terminal repeats. This extra DNA was more precisely mapped entirely within the EcoRI fragments D and d of SA-2, which were found to form part of the SmaI fragments U and P of SA-2 and Q and b of SA-2 and to contain one SmaI restriction enzyme site.     

Methods of measuring susceptibility of anaerobic bacteria to trovafloxacin, including quality control parameters

Three methods approved by the National Committee for Clinical Laboratory Standards for testing the susceptibility of anaerobic bacteria were used to evaluate the fluoroquinolone, trovafloxacin. The methods gave essentially comparable results with 126 anaerobes and with three quality control strains. A collaborative study defined the quality control range for trovafloxacin MICs. Trovafloxacin had good in vitro activity against the more common anaerobes (MIC 90 < = or 2.0 micrograms/ml).     

Rapid assessment of drug abuse in Ethiopia

A study of drug and substance abuse at Addis Ababa and in 24 towns across Ethiopia was conducted from June to November 1995. Five categories of respondents were selected for the study: street children, commercial sex workers and street vendors; medical, social and public health workers; law enforcement officials; leaders of religious institutions and educational establishments, youth leaders and personnel of non-governmental organizations providing social service to communities; and focus groups comprised of men and women from the various places covered in the study. All categories of respondents agreed that the problem of substance abuse was becoming increasingly serious in Ethiopia; that adolescents and young adults were the most affected group; and that addictive substances were easily obtainable in the country. The study also found that there was a significant increase in the number of Ethiopians chewing khat (Catha edulis). Khat, previously known to grow mainly in the eastern part of Ethiopia, was widely cultivated in all parts of the country. Khat consumption, traditionally confined to a certain segment of the population, had become popular among all segments of the population. Some of the respondents reported that khat-chewing often led to the abuse of illicit substances.     

Percutaneous balloon pericardiotomy for patients with recurrent pericardial effusion: using a novel double-balloon technique with one long and one short balloon

Percutaneous balloon pericardiotomy is effective and less invasive for the treatment of recurrent pericardial effusion. This study suggests that the double-balloon method with 1 longer and 1 shorter balloon is the procedure of choice for percutaneous balloon pericardiotomy.     

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.