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The paper presents the block diagram of the unit, describes some assemblies and its operation as a whole, gives information on the software and technical data. The computer-based EKS-K 1200 unit integrates a pacemaker, a 12-channel electrocardiograph with a monitor, and a database.
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This cross-sectional study was designed to assess differences in prevalence of contact dermatitis between machine operators exposed to metal-working fluids (MWFs) and unexposed assemblers, and to assess potential risk factors for contact dermatitis among these machine operators. In their work, machine operators were exposed to either semisynthetic or soluble oil MWFs. We evaluated 158 machine operators and 51 assemblers from one large automobile transmission plant using questionnaires, dermatologist examination of the skin, and dermal dosimetry to measure wetness and metal exposures. We found that machine operators had more combined (definite plus possible) dermatitis (27.2% vs. 13.7%, chi(2) = 3.9, p = 0.05, 1 df) compared with assemblers. Among machine operators, risk factors significantly associated with (combined) dermatitis were subjective assessment of wetness of the work, exposure to semisynthetic as opposed to soluble oil MWF, current cigarette smoking, and increasing worker age. These risk factors suggested preventive and control measures including control of wet work, surveillance program including early self-report of dermatitis, consideration of replacement of semisynthetic with soluble oil MWFs, and strictly limiting smoking among machine operators exposed to MWFs.
Yersiniabactin synthetase comprises four proteins, YbtE, HMWP1, HMWP2, and YbtU, encompassing seventeen functional domains, twelve catalytic and five carrier, to select, activate, and incorporate salicylate, three cysteines, and one malonyl moiety into the iron chelator yersiniabactin (Ybt). In the present study, yersiniabactin has been reconstituted in vitro from the 4 protein assembly line by the use of eight biosynthetic precursors. The rate of one turnover, comprising 22 chemical operations performed by the assembly line to release the completed Ybt molecule, was determined at 1.4 min(-1). During the course of Ybt production, the elongating acyl-S-enzyme chain was shown to transfer across a nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) interprotein interface and then a PKS/NRPS intraprotein interface. This study on the Ybt synthetase assembly line represents the first complete in vitro reconstitution of a nonribosomal peptide/polyketide hybrid system.
Recognition of the lac operator by the lac repressor involves specific interactions between residues in the repressor's recognition helix and bases in the DNA major groove. Tyr17 and Gln18, at positions 1 and 2 in the lac repressor recognition helix, can be exchanged for other amino acids to generate mutant repressors that display altered specificity. We have solved the solution structure of a protein-DNA complex of an altered-specificity mutant lac headpiece in which Tyr17 and Gln18 were exchanged for valine and alanine, respectively, as found in the recognition helix of the gal repressor. As previously described by Lehming et al. (EMBO J. 1987, 6, 3145-3153), this altered-specificity mutant of the lac repressor recognizes a variant lac operator that is similar to the gal operator Oe. The mutant lac headpiece showed the predicted specificity and is also able to mimic the gal repressor by recognizing and bending the natural gal operator Oe. These structural data show that, while most of the anchoring points that help the lac headpiece to assemble on the lac operator were preserved, a different network of protein-DNA interactions connecting Ala17 and Val18 to bases in the DNA major groove drives the specificity towards the altered operator.
In the design, development, and manufacturing stage of industrial products, engineers usually focus on the problems caused by hardware or software, but pay less attention to problems caused by "human error," which may significantly affect system reliability and safety. Although operating procedures are strictly followed, human error still may occur occasionally. Among the influencing factors, the inappropriate design of standard operation procedure (SOP) or standard assembly procedure (SAP) is an important and latent reason for unexpected results found during human operation. To reduce the error probability and error effects of these unexpected behaviors in the industrial work process, overall evaluation of SOP or SAP quality has become an essential task. The human error criticality analysis (HECA) method was developed to identify the potentially critical problems caused by human error in the human operation system. This method performs task analysis on the basis of operation procedure. For example, SOP, analyzes the human error probability (HEP) for each human operation step, and assesses its error effects to the whole system. The results of the analysis will show the interrelationship that exists between critical human tasks, critical human error modes, and human reliability information of the human operation system. To identify the robustness of the model, a case study of initiator assembly tasks was conducted. Results show that the HECA method is practicable in evaluating the operation procedure, and the information is valuable in identifying the means to upgrade human reliability and system safety for human tasks.
One of the important unsolved problems in the ribosome field is the molecular basis for the sequential and co-operative nature of ribosome assembly. As an approach to this problem, we have taken advantage of the temperature dependence of in vitro reconstitution and have used chemical probing methods to examine the conformation and reactivity of 16 S rRNA at successive stages during subunit assembly. One class of nucleotides displays reactivities similar to those observed in native 30 S particles when the RNA and protein are incubated in the absence of any heat step (0 degrees C effects). At 30 degrees C, where the assembly process takes 2 hours, other bases can be assigned to one of several additional kinetic classes, determined by the rate at which their chemical reactivities transit from levels observed in naked RNA to levels observed in fully assembled subunits: (1) fast (t1/2 = < 5 min at 30 degrees C); (2) slow (t1/2 = 15 to 30 min at 30 degrees C); (3) delayed slow (t1/2 = 30 to 60 min at 30 degrees C). Finally, several nucleotides display transient kinetics in their reactivities, showing increasing reactivity at early time points and becoming protected later in assembly; most of these effects correspond to residues that were previously shown to display reciprocal enhancement and protection patterns during step-wise in vitro assembly. These findings, together with our previous studies using purified individual proteins lead to the following conclusions: (1) there is a predominant 5' to 3' polarity to in vitro assembly, even though it is uncoupled from transcription; (2) portions of the central and 3' major domains fold into an active conformation only at a very late stage of assembly; (3) bases footprinted by late-assembling proteins, according to the 30 S subunit assembly map, show generally slower kinetics than residues footprinted by proteins that bind early in the assembly map, providing direct evidence for the sequential nature of the in vitro assembly process; (4) most proteins are associated with nucleotides that fall into more than one kinetic class, suggesting that assembly proceeds through multiple pathways, or that individual proteins interact sequentially with different regions of the RNA.
A practical method for assessing vibration exposure for workers operating vibrating hand tools on an automobile assembly line is presented. Vibration exposure is difficult to assess directly using many fast Fourier transform (FFT) spectral analyzers because of long task cycle times. Exposure time cannot be accurately estimated using time standards because of the high variability between operators and work methods. Furthermore, because workers frequently move about and get into inaccessible spaces, it is difficult to record vibration without interfering with the operation. A work sampling method was used for determining vibration exposure time by attaching accelerometers to the tools and suspending a battery-operated digital data logger from the air hose. Vibration acceleration and frequency spectra for each tool were obtained off-line replicating actual working conditions and analyzed together with exposure time data for determining individual worker vibration exposure. Eight pneumatic vibrating power hand tools, representing tools commonly used in an automobile assembly plant, were studied. Spectra for the rotary and reciprocating power tools and had large distinct dominant fundamental frequencies occurring in a narrow frequency range between 35 Hz and 150 Hz. These frequencies corresponded closely to tool free-running speeds, suggesting that major spectral component frequencies may be predicted on the basis of speed for some tools.
FtsZ is the major cytoskeletal protein operating in bacterial cell division. FtsZ assembles into protofilaments in vitro, and there has been some controversy over whether the assembly is isodesmic or cooperative. Assembly has been assayed previously by sedimentation and light scattering. However, these techniques will under-report small polymers. We have now produced a mutant of Escherichia coli FtsZ, L68W, which gives a 250% increase in tryptophan fluorescence upon polymerization. This provides a real-time assay of polymer that is directly proportional to the concentration of subunit interfaces. FtsZ-L68W is functional for cell division, and should therefore be a valid model for studying the thermodynamics and kinetics of FtsZ assembly. We assayed assembly at pH 7.7 and pH 6.5, in 2.5 mM EDTA. EDTA blocks GTP hydrolysis and should give an assembly reaction that is not complicated by the irreversible hydrolysis step. Assembly kinetics was determined with a stopped-flow device for a range of FtsZ concentrations. When assembly was initiated by adding 0.2 mM GTP, fluorescence increase showed a lag, followed by nucleation, elongation, and a plateau. The assembly curves were fit to a cooperative mechanism that included a monomer activation step, a weak dimer nucleus, and elongation. Fragmentation was absent in the model, another characteristic of cooperative assembly. We are left with an enigma: how can the FtsZ protofilament, which appears to be one-subunit thick, assemble with apparent cooperativity?
A description is given of the design and performance of a microcomputer-controlled respiratory servo system that incorporates the characteristics of a mechanical ventilator and also allows the performance of a multitude of test procedures required for assessment of pulmonary function in paralyzed animals. The device consists of a hydraulically operated cylinder-piston assembly and solenoid valves that direct inspiratory and expiratory gas flow and also enable switching to different test gas sources. The system operates as a volume-flow-preset ventilator but may be switched to other operational cycling modes. Gas flow rates may be constant or variable. The system operates as an assister-controller and, combined with a gas analyzer, can function as a "demand" ventilator allowing for set-point control of end-tidal PCO2 and PO2. Complex breathing maneuvers for a variety of single- and multiple-breath lung function tests are automatically performed. Because of the flexibility in selection and timing of respiratory parameters, the system is particularly suitable for respiratory gas studies.
The study was undertaken with the objective of observing operational aspects of 609 pulse polio booths established during Intensified Pulse Polio Campaign in East Delhi on National Immunisation Days (NID) during 2001-2002 through well designed format. The average number of persons present on duty at polio booths was 3.73 against a recommendation of 4 per booth. All the booths had vaccine carriers but some of them were not following the guidelines laid for maintaining the cold chain. One sixth "(15.4%)" of the booths needed fresh supplies of vaccine carrier. There is need for deployment of motivated trained manpower at polio booths to ensure the success of Pulse Polio Campaign. Maintenance of cold chain and quality assurance of vaccine administered to children in the field remain a challenge. More than half (55.7%) booths did not have any volunteer. Absence of volunteers from the community in more than half of the booths warrant for strengthening the linkages between the governmental agencies and the community.
In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.
Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes. It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11-15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.
Toxoplasma gondii infection is associated with intestinal microbiome disruption, but its effects on genome-resolved bacterial populations, the gut virome, and bacteriome-virome relationships remain poorly understood. Using previously generated shotgun metagenomic datasets from 36 intestinal samples collected from 18 Sprague-Dawley rats across control, acute, and chronic infection groups, we reconstructed 294 quality-filtered, non-redundant bacterial metagenome-assembled genomes (MAGs) and identified 899 medium-to-high-quality viral operational taxonomic units (vOTUs) from assembled metagenomic contigs. Infection was associated with reduced bacterial richness in the small intestine during both acute and chronic stages and lower Shannon diversity during chronic infection. In contrast, large-intestinal α-diversity remained stable despite significant compositional reorganization. Taxonomic changes included increased Lactobacillus intestinalis, Limosilactobacillus reuteri, and Prevotella sp900547005, together with decreased Rothia sp002492045 and Akkermansia muciniphila. Functional profiling revealed region- and stage-specific changes in predicted bacterial metabolic potential, including reduced energy-related pathways and carbohydrate-active enzyme abundance. The virome also showed significant compositional changes in both intestinal regions. Quimbyviridae and Podoviridae_crAss-like viruses decreased in the small intestine during chronic infection, while Quimbyviridae, Flandersviridae, and Podoviridae_crAss-like viruses showed stage-specific decreases in the large intestine. Predicted bacterial hosts were assigned to 48.39% of vOTUs, with Lachnospiraceae and Ruminococcaceae being the most frequently linked families. Trans-kingdom networks further revealed region-specific positive and negative abundance correlations between bacterial and viral taxa. These findings extend previous microbiota-metabolome observations by integrating genome-resolved bacteriome analysis with contig-based virome profiling, providing a foundation for future mechanistic studies of toxoplasmosis-associated microbiome remodeling.
We describe a manually operated system, simple to assemble, that will enable xenon-133 lung ventilation studies to be performed on ventilator-assisted patients. It can be constructed from items readily available. Radiation exposure levels to the operator were measured and are reported.
In 1988-1994 at the Second Surgical Clinic of the Masaryk University in Brno and at the Surgical Department of the Hospital of the Merciful Brethren in Valtice a total of 1037 diagnostic and therapeutic operations of the papilla of Vater were performed. Eventually after practice was assembled, the rate of successful operations was 94-99%. The authors emphasize the necessity to introduce endoscopic methods into the common practice of surgical departments; they emphasize the necessity of high-standard training of doctors and nurses. As compared with data from the literature and from other departments, the morbidity and mortality rate recorded by the authors was substantially lower.
We have previously reported the initial characterization of a catabolic operator site (O[rocA]) for the Bacillus subtilis arginine repressor/activator protein AhrC. Here, we present the characterization by gel retardation and DNase I footprinting of both O(rocA) and a second catabolic operator site, O(rocD). Both operator sites encompass a single recognition site, an ARG box, located immediately upstream of the transcriptional start points, a unique positioning for a transcriptional activator protein. Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around twofold, in their apparent affinities for the protein (K'd approximately 90 nM for O[rocA] and approximtaely 190nM for O[rocD]). This difference may result from the lower match to the ARG box consensus of the O(rocD) site. Both catabolic operators show evidence for co-operative binding with respect to protein concentration. Determination of the sequences of two AhrC catabolic operator sites, in combination with the three such biosynthetic sites, has allowed the derivation of an improved B. subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t. This is not identical to the Escherichia coli consensus operator for the AhrC homologue, ArgR, which may explain the only partial cross-functioning of these proteins in vivo. The O(rocA) site is adjacent to a sharp, stable bend located 5' to the catabolic operator. Circular permutation analysis has been used to determine the relative angle of bend (approximately 50 degrees), its location and the effect of adding magnesium ions and/or AhrC protein. Protein binding increases the relative bend angle to approximately 85 degrees. Bending is shown to be associated with a number of A-tracts in the upstream sequence. However, altering the phasing of the A-tracts has little effect on the affinity for AhrC. Truncation and competition experiments have been used to investigate the possible role of sequences flanking the operator on affinity. Very surprisingly, the affinity of the O(rocA) site appears to increase in the presence of excess, specific competitor fragment, i.e. the system shows anti-competitive effects. Competition is restored at high molar excesses of specific fragment over the protein. We propose a novel model for the assembly of a higher affinity form of AhrC at operator sites that is consistent with both the apparent co-operativity of binding and the anti-competitive effects. These data suggest that the molecular interactions occurring between the prokaryotic arginine-regulatory proteins and their operators may be more complex than is generally appreciated.
The kinetics of coupling of protein dimerization and DNA binding have been investigated in the biotin repressor system. Two repressor monomers bind to the 40 base-pair biotin operator sequence. In previous analyses of equilibrium-binding data the weak dimerization of the repressor has justified using a model in which two protein monomers bind cooperatively to the operator site. Here, rapid kinetic methods have been used to directly determine the binding mechanism. Results of rapid-mixing DNaseI footprinting measurements of association of the repressor with operator indicate that the binding process involves at least two steps. Results of measurements of the unimolecular dissociation of the complex reveal a half-life of approximately 400 seconds. Analysis of the data using a combination of simulation and global non-linear least-squares analysis provides support for a binding model in which a preformed repressor dimer associates with the biotin operator. This kinetic model is consistent with the previously proposed model for regulation of the functional switch in the repressor from enzyme to site-specific DNA-binding protein.