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SIR: My sometime colleague Imre Lakatos, the Popperian philosopher of science, had two self-explanatory terms to characterise bad science: 'monster barring' was one, and 'concept stretching' was the other. Quadrant [...]

The metropolitan Orlando industrial market experienced both good and bad news during 1993 and struggled to balance the mixed results and maintain optimism. The bulk distribution market was weakened when [...]

Bacterial cells undergo lysis readily, when suspended in mild aqueous acids, and release the cellular proteins along with other biomolecules. Molecular masses of the protein biomarkers released in-situfrom individual intact bacterial cells could be directly measured by mass spectrometry. Limited sample clean up may be required at times, prior to mass spectral analysis, to remove any ionizable impurities such as salts, buffers and deergents. The marker proteins specific for individual genus, species and strains were determined by the comparison of the biomarkers measured for several closely related organisms. Even though there is a probability of over 4000 cellular proteins expressed in any single bacterial cell, only a small fraction of the projected marker proteins are identified consistently during the process. This could be due to the variation in the ionization properties of the proteins and the limited energy available to prompt their ionization. Variation in the sample processing and culture conditions had little effect in the marker proteins observed during the process. This experimental procedure enables the distinction of gram positive as well as gram negative cellular pathogens and their corresponding non-pathogenic counterparts. The identity of few bacterial cells present in unknown samples can be easily, rapidly and accurately established by adopting a procedure involving simple sample processing followed by direct mass spectral analysis and data processing. Thus, an uncomplicated approach has been developed to resolve a complex problem involving cellular pathogens. This method has enormous application potential in the rapid identification and subsequent prevention of any potential health hazard caused by the pathogenic bacteria, either under natural or induced conditions. There is a great potential for the total automation of the entire process in the future for simpler but more effective unattended operations in the laboratory as well as in the field.

It has been proposed that distinct tertiary structures of the C-terminus of CCK-8 and CCK-58 result in differences in stimulation of pancreatic amylase secretion. Binding of CCK-8 and CCK-58 to CCK-A and CCK-B receptors and stability to enzymatic digestion were used as independent probes for tertiary structure of the C-terminus.

Cellular proteins (biomarkers) specific to any individual microorganism, determined by the direct mass spectral analysis of the corresponding intact cellular suspension, can be applied for the rapid and specific identification of the organisms present in unknown samples. The components of the bacterial suspensions, after a rapid separation over a C18 reversed-phase microcapillary column, were directly subjected to on-line electrospray ionization followed by analysis using an ion trap tandem mass spectrometer. This approach is equally effective for gram-positive as well as gram-negative bacteria but has a distinct advantage over our earlier reported method involving matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). During electrospray ionitation mass spectrometry (ESI-MS), liquid samples can be directly analyzed and there is the potential for developing tandem mass spectral methods for more specific identification of the individual organisms present in crude bacterial mixtures. The total analysis time leading to unambiguous bacterial identification in samples was less than 10 minutes and the results were quite reproducible. Miniaturization of the instrumentation along with total automation of this simple process could have immense impact on field operations. Routine, rapid, cost-effective field monitoring of environmental samples, agricultural products, samples from food processing, industrial sites and health institutions for suspected bacterial contamination could be a reality in the near future. Potential utility in biological, medical, bioprocessing, pharmaceutical, and other industrial research is also enormous. Copyright © 1999 John Wiley & Sons, Ltd.

ABSTRACTHepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.

The cathedral of Clermont exists as an interloper of the northern French Gothic style in a region long faithful to the Romanesque tradition. Without local stylistic antecedent, it appears without warning as an example of the mature Rayonnant style in an area where the preceding phases of Gothic architecture virtually had been ignored. Robert Branner's recent hypothesis, which postpones the beginning of construction from 1248 to 1262-1263, is important for the analysis of the cathedral's chronology, and its place within and impact upon the general development of Gothic architecture in the second half of the 13th century. Is the cathedral choir a monument which took shape in the late 1240s to the 1260s and logically builds upon the premises of Amiens, Reims, Beauvais, and the Paris transepts? Or is it, as Branner states, a tardy example of the restrained Court Style architecture of the mid-13th century? A careful analysis of the surviving documentation, corroborated by a study of the building itself, yields the conclusion that the choir of the cathedral of Clermont was constructed between about 1248 and 1280. Through a careful study of the plan and the fabric of the cathedral, the sections of the choir which were erected under the supervision of Jean Deschamps may be isolated. Though Deschamps can be credited only with the hemicycle and parts of the forechoir of Clermont, his work was followed scrupulously by his successors. This examination of Clermont and the work of Jean Deschamps sheds new light on the building history of a cathedral which, serving as a model for many of the major Gothic structures of the Midi, takes an important first step in defining the career of one of the key figures in French Gothic architecture of the second half of the 13th century.

The final phase of construction, begun in 1296, of Notre-Dame, the cathedral of Paris, encompassed sixteen chevet chapels, the enclosing wall of the liturgical choir, and an urgent campaign of structural consolidation. This study proposes that through the integration of a visually complex architecture with imagery, the master masons Pierre de Chelles and Jehan Ravy composed an environment that must be understood in terms of contemporary optical theory and devotional practices rather than modern notions of stylistic progress. Setting Notre-Dame within the context of fourteenth-century building in Paris illuminates the process of meaningful selection that guided its design.

The structural characterization of proteins and peptides isolated in minute quantities requires the most efficient use of available sample. A mass spectrometer data system was programmed to continuously evaluate incoming liquid chromatography/mass spectrometry data against a user-defined array of information. The resulting conclusions were used to automatically set and modify acquisition parameters in real time to collect collision-induced dissociation spectra for selected ions (tandem mass spectrometry). This approach has provided a mechanism to target specific subsets of masses in a complex mixture and/or to discriminate selectively against masses that are known or not of interest. Masses of contaminants or peptide masses derived from known proteins can be automatically recorded and removed from further consideration for collision-induced dissociation analysis. Once recorded, these “libraries” of masses can be used across multiple analyses. This technique directs the mass spectrometer data system to focus on the analysis of masses significant to the user, even if their signal intensities are well below the intensities of contaminating masses. When combined with a database search program to correlate tandem mass spectra to known protein sequences, the identity of the protein can be established unequivocally by using less than 100 fmol of sample.

A microscale, variable flow, liquid chromatography-electrospray ionization source has been coupled to an ion trap mass spectrometer and used to analyze both simple and complex protein mixtures. By using an analytical technique described as “peak parking,” the sample analysis times for components eluting from a liquid Chromatograph can be greatly increased without sacrificing Chromatographic resolution. This was achieved by instantaneously reducing the column head pressure (and hence, flowrate) under peaks of interest to prolong their elution from the tip. With the technique, it is possible to perform manual parent ion selection, higher resolution narrow mass range scans, and multiple stages of tandem mass spectrometry (MS2and MS3) within the context of a single eluting peak. Benefits normally associated with an off-line tandem mass spectrometry analysis, such as the optimization of collision induced dissociation parameters and the analysis of more than one charge state, can now be obtained with the increased sensitivity and selectivity afforded by the chromatography. The utility of this method for the comprehensive analysis of complex mixtures was illustrated with the analysis of an in-gel protein digest mixture derived from a single spot from a two-dimensional electrophoresis gel. In addition to the expected enzyme digestion products, other peptides derived from nonspecific cleavage by the enzyme and protein impurities, as well as those having oxidized, derivatized, or deamidated amino acid residues were fully characterized.

A methodology has been developed for the rapid identification of gel separated proteins. Following in gel protein digestion with trypsin, the resulting peptide mixture is analyzed by on-line liquid chromatography, electrospray mass spectrometry (LC/MS). The mass spectral data containing either accurate mass values or sequence specific fragment ion information is then matched to a database of known protein sequences. Key features of the LC/MS system are the use of a novel integrated, microscale LC column-electrospray interface and variable flow solvent delivery to optimize the efficiency of sample loading and gradient elution. With these enhancements, only 10 min is required to analyze each sample. The method is routine for sample amounts ranging from 50 to 500 fmol. The analysis parameters for the ion trap mass spectrometer have to be carefully adjusted in order to keep pace with the rapidly eluting LC peaks. Although designed for rapid LC separations, the integrated column-electrospray interface is also able to provide extended analyses of selected components using a technique known as “peak parking. ”

The functionality of fuel cell vehicles is continuously being improved by several automakers and efforts are being made to demonstrate performance, durability, efficiency and freeze start capability comparable to conventional vehicles. Recent results demonstrating acceptable operational performance, efficiency, freeze start capability and durability are shown and discussed. The main barrier preventing fuel cells from being competitive with conventional ICE is the high cost of the stack and system. For the stack, the key cost driver is catalyst loading. Requirements for future catalysts will be presented, demonstrating the challenges and technology gaps following from a reduction of catalyst loadings as well as the use of PtCo based catalysts.

Several letters to the editor are presented in response to the articles in previous issues including "How to Rethink Arts Funding," by Michael Connor in the October 2008 issue, "Our Misgoverned Universities," by Mr. James Allan in the October 2008 issue and "Nude or Naked," by Patricia Anderson in the September 2008 issue.