Molecular basis of reduced glucosylceramidase activity in the most common Gaucher disease mutant, N370S

Gaucher disease is caused by the defective activity of the lysosomal hydrolase, glucosylceramidase. Although the x-ray structure of wild type glucosylceramidase has been resolved, little is known about the structural features of any of the >200 mutations. Various treatments for Gaucher disease are available, including enzyme replacement and chaperone therapies. The latter involves binding of competitive inhibitors at the active site to enable correct folding and transport of the mutant enzyme to the lysosome. We now use molecular dynamics, a set of structural analysis tools, and several statistical methods to determine the flexible behavior of the N370S Gaucher mutant at various pH values, with and without binding the chaperone, N-butyl-deoxynojirimycin. We focus on the effect of the chaperone on the whole protein, on the active site, and on three important structural loops, and we demonstrate how the chaperone modifies the behavior of N370S in such a way that it becomes more active at lysosomal pH. Our results suggest a mechanism whereby the binding of N-butyl-deoxynojirimycin helps target correctly folded glucosylceramidase to the lysosome, contributes to binding with saposin C, and explains the initiation of the substrate-enzyme complex. Such analysis provides a new framework for determination of the structure of other Gaucher disease mutants and suggests new approaches for rational drug design.     

A single cyanobacterial ribotype is associated with both red and black bands on diseased corals from Palau

Filamentous cyanobacteria forming red and black bands (black band disease, BBD) on 3 scleractinian corals from Palau were molecularly identified as belonging to a single ribotype. Red cyanobacterial mats sampled from infections on Pachyseris speciosa and a massive Porites sp. yielded red strains RMS1 and RMS2 respectively; the black cyanobacterial mat sampled from an infection on Montipora sp. yielded black strain BMS1. Following trials of a range of specialized media and culture conditions, 2 media, Grund and ASN-III, were identified as the best for successful isolation and culturing. Cultured cyanobacteria were examined under a light microscope to establish purity, color and morphological appearance. DNA extraction and partial sequencing of the 16S rDNA gene of both red and black cyanobacterial isolates demonstrated 100% sequence identity. These isolated strains were also found to have 99% sequence identity with an uncultured cyanobacterial strain previously identified by molecular techniques as belonging to a cyanobacterial ribotype associated with BBD-infected corals in the Caribbean. This is the first report of the successful isolation and culture of cyanobacterial strains derived from both red bands and BBD. Based on these findings, it is suggested that the classification of these 2 syndromes as separate coral diseases be postponed until further evidence is collected.     

Adenine-guanine base pairing ribosomal RNA.

Analyses of secondary structures proposed for ribosomal RNA's show that, of the different kinds of base pairs directly adjoining the ends of postulated double-helical regions, only A-G with A at the 5' end significantly exceeds the number expected for a random base distribution. An A(syn)-G(trans) hydrogen-bonded basepair is proposed. This could fit at the end of an undistorted double helix, but would prevent further base stacking, thus favoring a break in the double helix to produce a non-linear tertiary structure.     

Integrin shedding as a mechanism of cellular adaptation during cardiac growth

Integrin-mediated cell-extracellular matrix (ECM) interactions are essential for multiple cellular processes; however, little is known regarding integrin turnover during these events. Recent studies have demonstrated shedding of cell surface molecules and suggested this as a potential mechanism for integrin turnover. Confocal microscopy of mouse hearts under different physiological conditions demonstrated the presence of beta(1)-integrin-immunoreactive material in the interstitium. Culture media from neonatal rat cardiac myocytes and fibroblasts contained a 55-kDa fragment of beta(1)-integrin. Attachment to ECM components, response to phorbol 12-myristate 13-acetate stimulation, and matrix metalloproteinase inhibition assays demonstrated that fibroblasts responded differently to the fragment compared with myocytes. The beta(1)-integrin fragment stimulated myocyte attachment to collagen and the fragment itself bound a variety of ECM proteins. These studies indicate that as myocytes and fibroblasts change size and shape, cellular contacts with the ECM are altered, resulting in the liberation of a beta(1)-integrin fragment from the cell surface. Integrin shedding may represent a novel mechanism of rapidly modifying cell-ECM contacts during various cellular processes.     

A difference in the regulation of mRNA expression between the phenotypic and the embryonic alkaline phosphatase genes in human cancer cells

The steady-state levels of mRNAs encoding alkaline phosphatase isoenzymes were examined in two human breast carcinoma cell lines. MDA-MB-157 cells expressed the phenotypic breast alkaline phosphatase and BT20 cells expressed the nonphenotypic placental alkaline phosphatase isoenzyme, frequently reexpressed in neoplasms. Dexamethasone (DEX), which elicits a general effect on phosphatase expression, and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), a promoter of cell differentiation that correspondingly effects embryonic phosphatase expression, were chosen as perturbing agents for these experiments. RNA blot analysis showed a single RNA species of approximately 2.6 kb under all treatment conditions in BT20 cells and a single RNA species of 2.6 kb under each condition in MDA-MB-157 cells. The results showed that the expression of both the AP isoenzyme mRNA phenotypic of breast produced by MDA-MB-157 cells and the embryonic alkaline phosphatase isoenzyme (PLAP) mRNA produced by BT20 cells was increased by treatment with DEX. By comparison 1,25(OH)2D3 caused an increase in the tissue-unspecific AP mRNA in the MDA-MB-157 cells, but caused a decrease in PLAP mRNA levels in BT20 cells. The level of each isoenzyme mRNA species is altered by either hormone in a dose- and time-dependent manner in both cell lines. In BT20 cells, treatment with cycloheximide showed that ongoing protein synthesis is not required to potentiate the PLAP mRNA response to DEX, but is required for the action of 1,25(OH)2D3. However, protein synthesis is required for the action of both hormones in the MDA-MB-157 cells which make the breast phenotypic AP. These data demonstrate that the DEX- and 1,25(OH)2D3-regulated expression of both of these alkaline phosphatase isoenzymes occurs via a complex mechanism involving control of mRNA abundance, not translational control of constant message levels.     

Solvent accessibility as a predictive tool for the free energy of inhibitor binding to the HIV-1 protease.

We have developed a simple approach for the evaluation of the free energies of inhibitor binding to the protease of the human immunodeficiency virus (HIV-1 PR). Our algorithm is based on the observation that most groups that line the binding pockets of this enzyme are hydrophobic in nature. Based on this fact, we have likened the binding of an inhibitor to this enzyme to its transfer from water to a medium of lower polarity. The resulting expression produced values for the free energy of binding of inhibitors to the HIV-1 PR that are in good agreement with experimental values. The additive nature of this approach has enabled us to partition the free energy of binding into the contributions of single fragments. The resulting analysis clearly indicates the existence of a ranking in the participation of the enzyme's subsites in binding. Although all the enzyme's pockets contribute to binding, the ones that bind the P2-P'2 span of the inhibitor are in general the most critical for high inhibitor potency. Moreover, our method has allowed us to determine the nature of the functional groups that fit into given enzyme binding pockets. Perusal of the energy contributions of single side chains has shown that a large number of hydrophobic and aromatic groups located in the central portion of the HIV-1 PR inhibitors present optimal binding. All of these observations are in agreement with experimental evidence, providing a validation for the physical relevancy of our model.     

Magnetic field exposure among utility workers

The Electric and Magnetic Field Measurement Project for Utilities—the Electric Power Research Institute (EPRI) Electric and Magnetic Field Digital Exposure (EMDEX) Project (the EPRI EMDEX Project)—was a multifaceted project that entailed technology transfer, measurement protocol design, data management, and exposure assessment analyses. This paper addresses one specific objective of the project: the collection, analysis, and documentation of power-frequency magnetic field exposures for a diverse population of utility workers. Field exposure data measured by an EMDEX system were collected by volunteer utility employees at 59 sites in four countries between September, 1988, and September, 1989. Specially designed sampling procedures and data collection protocols were used to ensure uniform implementation across sites. Volunteers within 13 job classifications recorded which of eight work or three nonwork environments they occupied while wearing an EMDEX meter. Approximately 50,000 hours of magnetic field exposure records taken at 10 s intervals were obtained, about 70% of which were from work environments. Exposures and time spent in environments were analyzed by primary work environment, by occupied environment, and by job classification. Generally, for utility-specific job classifications related to the generation, transmission, and distribution of electricity, the field and exposure measurements in terms of workday mean field were higher than in more general occupations. The job classifications with the highest (median workday mean) exposure were substation operators (0.7 μT) and electricians (0.5μT). Total variance also tended to be largest for utility-specific job classifications. For these workers, the contributions of between-worker and within-worker variances to total variance were about the same. Measurements in utility-specific environments were higher than in more general environments. Estimates of time-integrated exposure indicated that utility-specific job classifications received about one-half or more of their total exposure on the job. The nonwork field and exposure distributions for workers in all job categories were comparable with median nonworkday means of about 0.09 μT. © 1995 Wiley-Liss, Inc.     

A survey of the distribution and density of the primates of Guyana

In January through February 1994, we conducted the first broad-scale survey of Guyanese primates since 1975. Our goals were (1) to follow up questions raised in the earlier survey, (2) to compare population densities, and (3) to locate potential sites for future long-term research. We used distributional survey methods along trails and rivers and interviewed local inhabitants in each region. We surveyed five general areas, two of which had been studied in 1975. The distribution reported in 1975 for five monkey species—Alouatta seniculus, Cebus olivaceus, Pithecia pithecia, Chiropotes satanas,and Saimiri sciureus—was confirmed. However, questions were raised concerning the western extent of the range of three species: Ateles paniscus, Cebus apella,and Saguinus midas.In comparing densities between 1994 and 1975, we found a significant drop in group densities over the past 20 years and a shift in relative proportions of individual primate species over time. For example, although the total number of kilometers surveyed was identical, group densities were three times higher in 1975 than in 1994. Further, group densities of Ateles, Alouatta,and Pitheciawere much lower, while those of Saguinuswere similar in both years. These findings strongly suggest that habitat destruction and continued hunting pressure are affecting the primate populations.