Identifying sequence determinants of reduction potentials of metalloproteins

The reduction potential of an electron transfer protein is one of its most important functional characteristics. Although the type of redox site and the protein fold are the major determinants of the reduction potential of a redox-active protein, its amino acid sequence may tune the reduction potential as well. Thus, homologous proteins can often be divided into different classes, with each class characterized by a biological function and a reduction potential. Site-specific mutagenesis of the sequence determinants of the differences in the reduction potential between classes should change the reduction potential of a protein in one class to that of the other class. Here, a procedure is presented that combines energetic and bioinformatic analysis of homologous proteins to identify sequence determinants that are also good candidates for site-specific mutations, using the [4Fe–4S] ferredoxins and the [4Fe–4S] high-potential iron–sulfur proteins as examples. This procedure is designed to guide site-specific mutations or more computationally expensive studies, such as molecular dynamics simulations. To make the procedure more accessible to the general scientific community, it is being implemented into CHARMMing, a Web-based portal, with a library of density functional theory results for the redox site that are used in the setting up of Poisson–Boltzmann continuum electrostatics calculations for the protein energetics.     

Normal and transforming Ras are differently regulated for posttranslational modifications

Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly¹²) or the transforming (Val¹²) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter. Expression of either type of Ras was efficiently induced by the cadmium treatment of these cells, and immunoprecipitation of metabolically labeled cell extracts revealed that both normal and transforming Ras were expressed as four differently migrating forms on SDS-polyacrylamide gels, two of which were slower migrating cytosolic precursors and the other two were faster migrating membrane-bound forms. There was no significant difference in half lives between normal and transforming Ras; however, posttranslational modification was quite different between the two types of Ras. Transforming Ras was processed and became membrane-bound forms much more efficiently than normal Ras. Interestingly, posttranslational modification and membrane localization of Ras was significantly inhibited when the c-myc oncogene was co-expressed with Ras. In contrast to the c-myc oncogene, expression of either wild type or mutant p53 did not affect the posttranslational modification of Ras, suggesting that the c-myc oncogene specifically impairs the posttranslational modification of Ras. © 1996 Wiley-Liss, Inc.     

Quantitation using a stable isotope dilution assay (SIDA) and thresholds of taste-active pyroglutamyl decapeptide ethyl esters (PGDPEs) in sake

A stable isotope dilution assay (SIDA) for two taste-active pyroglutamyl decapeptide ethyl esters (PGDPE1; (pGlu)LFGPNVNPWCOOC₂H₅, PGDPE2; (pGlu)LFNPSTNPWCOOC₂H₅) in sake was developed using deuterated isotopes and high-resolution mass spectrometry. Recognition thresholds of PGDPEs in sake were estimated as 3.8 μg/L for PGDPE1 and 8.1 μg/L for PGDPE2, evaluated using 11 student panelists aged in their twenties. Quantitated concentrations in 18 commercial sake samples ranged from 0 to 27 μg/L for PGDPE1 and from 0 to 202 μg/L for PGDPE2. The maximum levels of PGDPE1 and PGDPE2 in the sake samples were approximately 8 and 25 times higher than the estimated recognition thresholds, respectively. The results indicated that PGDPEs may play significant sensory roles in the sake. The level of PGDPEs in unpasteurized sake samples decreased during storage for 50 days at 6 °C, suggesting PGDPEs may be enzymatically decomposed.     

A familial case of visceral toxocariasis due to consumption of raw bovine liver

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.     

Laboratory appraisal of optimal gaseous conditions for growth of zoonotic Helicobacter felis ATCC 49179

An attempt was made to assess the hitherto undescribed optimal gaseous conditions for growth of zoonotic Helicobacter felis, focusing on the ratio of spiral-forms amongst the whole cells examined. The largest mean colony diameter was obtained under the gaseous condition of O₂ 12% and CO₂ 10%. In analyzing the five day old colonies, the highest percentage of spiral forms (85.5%) was observed under the condition of O₂ 18% and CO₂ 5%. In contrast, the lowest percentage of spiral forms (2.3%) was demonstrated under the condition of O₂ 1% and CO₂ 10%. The condition of O₂ 12% and CO₂ 10% was concluded to be optimal for obtaining cells with the largest colony sizes, although colonies proliferated under such conditions definitely contain many more coccoid cells than spiral forms. In culturing H. felis strains, optimal gaseous conditions should be employed according to the purposes or preferences of study designs.     

Notes on some plant-inhabiting fungi collected from the Nansei Islands (3)*

In this paper, four plant-inhabiting fungi newly collected from the Nansei Islands, Japan, are reported. Among them, Phaeodiscula tremae Sawada from Tokunoshima Island was transferred to the genus Capnodiastrum and given a new name C. sawadae, because in the genus Capnodiastrum, the specific epithet tremae has already been used for the different South American species. Nyssopsora formosana on Koelreuteria formosana and Pestalotiopsis fici on Ardisia quinquegona collected in Okinawa Main Island and Pseudocercospora acaciae-confusae on Acacia confusa collected in Okinoerabu Island were newly recorded from Japan.     

Fatigue and tensile properties of radicular dentin substrate

The purpose of this study was to compare the fatigue and tensile strengths of radicular dentin. Forty bovine lower central incisors were used, twenty teeth for the fatigue test and twenty teeth for the tensile test. Bovine teeth were each sectioned into coronal and radicular portions. Dentin slabs of 1mm thickness were prepared along the radicular tooth using a low-speed cutting machine and trimmed into dumbbell-shaped specimens. A dentin slab was harvested from each tooth. Subsequently, fatigue and tensile tests were performed in Hank's balanced saline solution at 37 °C. The staircase method was employed to determine fatigue strength and its standard deviation. Fracture surfaces were observed by scanning electron microscopy. Mean fatigue strength and tensile strength were 44.3±5.0 and 84.4±8.3 MPa, respectively. The fatigue strength of radicular dentin was significantly lower than the tensile strength. The fatigue strength of radicular dentin was only approximately one half of the tensile strength.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Measures of Autozygosity in Decline: Globalization, Urbanization, and Its Implications for Medical Genetics

This research investigates the influence of demographic factors on human genetic sub-structure. In our discovery cohort, we show significant demographic trends for decreasing autozygosity associated with population variation in chronological age. Autozygosity, the genomic signature of consanguinity, is identifiable on a genome-wide level as extended tracts of homozygosity. We identified an average of 28.6 tracts of extended homozygosity greater than 1 Mb in length in a representative population of 809 unrelated North Americans of European descent ranging in chronological age from 19–99 years old. These homozygous tracts made up a population average of 42 Mb of the genome corresponding to 1.6% of the entire genome, with each homozygous tract an average of 1.5 Mb in length. Runs of homozygosity are steadily decreasing in size and frequency as time progresses (linear regression, p<0.05). We also calculated inbreeding coefficients and showed a significant trend for population-wide increasing heterozygosity outside of linkage disequilibrium. We successfully replicated these associations in a demographically similar cohort comprised of a subgroup of 477 Baltimore Longitudinal Study of Aging participants. We also constructed statistical models showing predicted declining rates of autozygosity spanning the 20th century. These predictive models suggest a 14.0% decrease in the frequency of these runs of homozygosity and a 24.3% decrease in the percent of the genome in runs of homozygosity, as well as a 30.5% decrease in excess homozygosity based on the linkage pruned inbreeding coefficients. The trend for decreasing autozygosity due to panmixia and larger effective population sizes will likely affect the frequency of rare recessive genetic diseases in the future. Autozygosity has declined, and it seems it will continue doing so.