A system for mutation measurement in mammalian cells: application to gamma-irradiation

Monitoring of mutagenesis by environmental agents for the purpose of preventing genetic disease including cancer must include quantitation of cell killing, sensitive measurement of mutation production by appropriate doses of each agent, and assessment of mutation repair effects in mammalian cells. A four-step procedure, in the presence and absence of a repair suppressor, is proposed: (i) determination of the survival curve; (ii) measurement of the mitotic index in cells collected after treatment with colcemid; (iii) construction of a mutagenesis yield curve in the presence and absence of a repair suppressor, like caffeine; and (iv) assessment of the effect of test agents on the repair of mutations produced by other mutagens. The procedure is quantitative, reproducible, and reasonably rapid. It involves measurement of mutations causing visible chromosomal aberrations. Numerical parameters are proposed defining quantitatively mutation, cell killing, and mutation repair capacity. The procedure is applied to gamma-irradiation and can detect the effects of doses as low as 2-5 cGy. Theoretical analysis of the underlying processes is presented, using the concept of D(0)E, the effective dose of mutagen after repair mechanisms and neutralizing agents have acted. Microscopically visible chromosome aberrations are due to mutations that distort the process of mitotic chromosome condensation, with or without actual chromosome breakage.     

p53 gene mutation: software and database

Facial aging is a biological phenomenon. Skin properties change with time, and gravity and facial expressions exert mechanical deformation. Knowledge of these alterations may suggest ways to reverse them by identifying the corresponding distortional forces. The aim of this study was to determine a pattern of change for parameters of the face during the aging process, based on the numerical fitting of measures from a sample of patients. The first aspect of this study was to define adequate facial parameters and means of measuring them. Subsequently, each parameter was defined individually, and these data were analyzed as a set. The sample for the research was restricted to a group of 40 white female patients with a history of limited exposure to the sun, with ages ranging from 25 to 65. The reason for choosing this sample was the availability of frontal pattern photographs at different ages. The parameters for each patient were measured at two different ages. A strong correlation was found between age and behavior of the parameters. This aging model can be verified qualitatively by comparing photographs of a patient with manipulated photographs simulating aging. The quantitative verification of the model was done through the comparison of the measured and the predicted parameters.     

An Internet linkage and mutation database for the complex phenotype asthma

SUMMARY: The paper presents details of database construction, website installation and server architecture of the asthma and allergy gene database. AVAILABILITY: Database and server templates are available on request from the first author. SUPPLEMENTARY INFORMATION: The URL of the asthma and allergy gene database is http://cooke.gsf.de     

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.     

EpoDB: a prototype database for the analysis of genes expressed during vertebrate erythropoiesis

EpoDB is a database of genes expressed in vertebrate red blood cells. It is also a prototype for the creation of cell and tissue-specific databases from multiple external sources. The information in EpoDB obtained from GenBank, SWISS-PROT, Transfac, TRRD and GERD is curated to provide high quality data for sequence analysis aimed at understanding gene regulation during erythropoiesis. New protocols have been developed for data integration and updating entries. Using a BLAST-based algorithm, we have grouped GenBank entries representing the same gene together. This sequence similarity protocol was also used to identify new entries to be included in EpoDB. We have recently implemented our database in Sybase (relational tables) in addition to SICStus Prolog to provide us with greater flexibility in asking complex queries that utilize information from multiple sources. New additions to the public web site (http://www.cbil.upenn.edu/epodb) for accessing EpoDB are the ability to retrieve groups of entries representing different variants of the same gene and to retrieve gene expression data. The BLAST query has been enhanced by incorporating BLASTView, an interactive and graphical display of BLAST results. We have also enhanced the queries for retrieving sequence from specified genes by the addition of MEME, a motif discovery tool, to the integrated analysis tools which include CLUSTALW and TESS.     

Electrically generated silver ions: quantitative effects on bacterial and mammalian cells

The inhibitory and bactericidal concentrations of electrically generated silver ions were 10 to 100 times lower than for silver sulfadiazine. Effects on normal mammalian cells were minimal.     

A strategy for database interoperation

To realize the full potential of biological databases (DBs) requires more than the interactive, hypertext flavor of database interoperation that is now so popular in the bioinformatics community. Interoperation based on declarative queries to multiple network-accessible databases will support analyses and investigations that are orders of magnitude faster and more powerful than what can be accomplished through interactive navigation. I present a vision of the capabilities that a query-based interoperation infrastructure should provide, and identify assumptions underlying, and requirements of, this vision. I then propose an architecture for query-based interoperation that includes a number of novel components of an information infrastructure for molecular biology. These components include a knowledge base that describes relationships among the conceptualizations used in different biological databases, a module that can determine the DBs that are relevant to a particular query, a module that can translate a query and its results from one conceptualization to another, a collection of DB drivers that provide uniform physical access to different database management systems, a suite of translators that can interconvert among different database schema languages, and a database that describes the network location and access methods for biological databases. A number of the components are translators that bridge the heterogeneities that exist between biological DBs at several different levels, including the conceptual level, the data model, the query language, and data formats.     

Fourier-transform Raman spectra of ivory. III: Identification of mammalian specimens

The FT-Raman spectra of six mammalian ivories, other than elephant and mammoth, are presented and spectral differences formulated into a protocol for the identification of animal species from the ivory samples. In this study, sperm whale, walrus, wart hog, narwhal, hippopotamus and domestic pig are considered. The results, which are obtained non-destructively from a variety of specimens, suggest that FT-Raman spectroscopy provides a potentially useful method for the identification of mammalian ivory.     

Evolution of the mammalian Y chromosome and sex-determining genes

Fibrosis is characterized by proliferation of fibroblasts and deposition of extracellular matrix (ECM). As alterations in the composition of ECM may account for its chronic extension, we studied the expression of the tenascin-C (TN-C) and tenascin-X (TN-X) ECM glycoproteins in our pig model of the effects of accidental exposures to radiation, in which cutaneous and muscle fibrosis developed after the induction of necrosis after a high single dose (160 Gy at the skin surface) of gamma rays. We found that, in the healed fibrotic dermis and underlying muscle fibrosis, the amount of TN-C mRNA was increased up to 18- and 39-fold, respectively, compared to normal dermis, whereas the level of TN-X mRNA remained almost unchanged. In analyses by Western blotting, the two main TN-C isoforms of 235-240 and 190-200 kDa increased up to 45- and 105-fold in fibrotic tissues, respectively. The large isoform was expressed more strongly than the smaller, although in healed fibrotic scar tissues their ratio was lower in protein than in RNA. Compared to unirradiated skin, an immunohistological study revealed stronger TN-C staining at the dermo-epidermal junction and in areas of remodeling in healed skin. An intense extracellular staining was observed around myofibroblasts in muscle fibrosis. Therefore, the gene encoding TN-C is highly up-regulated in fibrotic tissues, and mechanisms regulating the levels of TN-C variants occur at both the RNA and protein levels. Each isoform might play a distinct role in the chronic activation of fibrosis by differentially regulating mechanisms like cell adhesion, migration or proliferation.     

A thermodynamic database for copper smelting and converting

The thermodynamic properties of the slag, matte, and liquid copper phases in the Cu-Ca-Fe-Si-O-S system have been critically assessed and optimized over the ranges of compositions of importance to copper smelting/converting based on thermodynamic and phase equilibria information available in the literature and using the modified quasichemical model. A thermodynamic database has been developed, which can be used for the calculation of matte-slag-copper-gas phase equilibria of interest for the production of copper. The model reproduces within experimental error limits all available experimental data on phase diagrams, matte-alloy miscibility gap and tie-lines, enthalpies of mixing, and activities of Cu and S in the matte and liquid alloy. The calculated solubilities of Cu in both S-free slag and slag equilibrated with matte are also in good agreement with experiment under all studied conditions, such as at SiO₂ saturation, in equilibrium with Fe, Cu, or Cu-Au alloys, at fixed oxygen or SO₂ partial pressures and at different contents of CaO in the slag. Sulfide contents (sulfide capacities) of the slags are predicted within experimental error limits from the modified Reddy-Blander model, with no adjustable parameters. As an example of the application of the database, the stability field of matte/slag equilibrium is calculated, and the matte and slag compositions are plotted vs iron to silica ratio in the slag at various SO₂ pressures over this field. The matte-slag two-phase field is limited by the calculated lines corresponding to precipitation of copper, silica, and magnetite.     

Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems

Engineered antibody molecules, and their fragments, are being increasingly exploited as scientific and clinical tools. However, one factor that can limit the applicability of this technology is the ability to express large amounts of active protein. In this review we describe the relative advantages and disadvantages of bacterial, yeast, insect and mammalian expression systems, and discuss some of the problems that can be encountered when using them. There is no 'universal' expression system, that can guarantee high yields of recombinant product, as every antibody-based molecule will pose its own problems in terms of expression. As a result the choice of system will depend on many factors, including the molecular species being expressed, the precise sequence of the individual antibody and the preferences of the individual investigator. However, there are general rules with regards to the design of expression vectors and systems which will help the investigator to make informed choices as to which strategy might be appropriate for their application.     

Modeling and measurement of the spontaneous mutation rate in mammalian cells

In order to examine the respiratory effects of tonic-clonic seizures and their treatment with i.v. diazepam or lorazepam, we utilized a spontaneously breathing piglet seizure model. A tracheostomy, arterial catheter, and epidural electrodes were inserted and pigs were maintained under ketamine anesthesia. After baseline recordings, seizures were induced with a pentylenetetrazol (PTZ) bolus and a 20 min infusion (5-6 mg/kg/min). After 10 min of PTZ infusion, randomly assigned animals received diazepam (D; N = 7; 0.5 mg/kg), lorazepam (L; N = 7; 0.2 mg/kg), or 0.9% saline (C; N = 7; controls) by rapid peripheral vein injection. Minute ventilation (Ve), Pa(CO2), and the pressure change in response to airway occlusion at end-expiration (P0.1) were measured at standard intervals. All groups had comparable increases in respiratory drive during untreated seizures. Changes in Ve and P0.1 were reduced to at or below baseline values in groups D and L, but not C, from 2 to 45 min after treatment (P < 0.05). No significant changes were observed in Pa(CO2) after either intervention. Following anticonvulsants, the cumulative duration of seizures was significantly reduced in L and D groups, compared to C (P < 0.05). We conclude that increases in respiratory drive occur during tonic-clonic seizures induced with PTZ. Amelioration of seizure activity with lorazepam or diazepam results in a reduction in respiratory drive, but not respiratory failure, in this tracheostomized model.     

Single nucleotide polymorphism spectra in newborns and centenarians: identification of genes coding for rise of mortal disease

Some single-nucleotide polymorphisms (SNPs) increase the risk of mortal disease. Identifying these SNPs and the genes in which they reside is an important area in human genomics. Such qualitative observations are important in themselves. However, an accurate assessment of the numerical distribution and age-dependent decline of SNPs in the population would permit calculation of the rises represented by each SNP. Such analyses have not been attempted because of a lack of an efficient and cost-effective method to detect multiple SNPs in a large number of individuals and a large number of genes. Here, we suggest the use of an analytical procedure that can scan for SNPs in 100-bp DNA sequences from as many as 10000 donors' blood cell samples, or 20000 alleles, simultaneously. Our suggestion is based on technology developed for studies of somatic mutations in human tissue DNA for point mutations at frequencies equal to or greater than 10(-6). In a simplified version of this technology, any SNP arising at frequencies at or above 5x10(-4) would be identified with useful precision. A gene would be represented by 10 or more sections of 100bp. This strategy includes splice-site mutations that represent a significant fraction of gene inactivating point mutations and would not be observed in strategies using cDNA. To illustrate the logic of the suggested approach, we use American mortality records to calculate the expected decrease in SNPs coding for premature mortality in newborns and centenarians. We consider several elementary cases: SNPs in one gene only, any of several genes, or all of several genes that create a risk of death by pancreatic cancer. The fraction of expressed polymorphisms affecting mortality should be simultaneously increased in probands and decreased in the aged relative to newborns. Silent polymorphisms in the same gene would remain unchanged in all three groups and serve as internal standards. A key point is that scanning a gene, in which loss of gene function creates the risk of mortality is expected to reveal not one, but multiple SNPs, which decline with age, as carriers die earlier in life than non-carriers. Several SNPs in a scanned gene would suggest that the decreasing SNP was genetically linked to a different polymorphism that creates the disease risk.     

Functions of mammalian Polycomb group and trithorax group related genes

OBJECTIVE: Norplant and Norplant-2 have been available for use by Finnish women since 1984 and 1986, respectively. The objective of this study was to explore Norplant users' experiences of insertions, removals and medical treatments. DESIGN: A questionnaire was sent to women who had received Norplant or Norplant-2 implants one to two years earlier (n = 262) in normal clinical settings; the response rate was 79%. RESULTS: At insertion, problems were experienced by 9% and at least some pain by 23% of women. During the first year 20% of all users (14% of Norplant and 33% of Norplant-2 users) had their implants removed. Problems were experienced at removal by 33% of women and pain was experienced by 40%; nevertheless most users were satisfied with the device. Findings were similar for the two types of Norplant. Forty-two percent of the women had received minipills containing levonorgestrel before Norplant insertions in an attempt to assess Norplant's suitability, and 8% had received drugs for adverse effects caused by Norplant. CONCLUSIONS: Studies including perspectives of Norplant users and the whole lifespan of Norplant (including removal) should be conducted in all clinical settings where Norplant is provided.     

An investigation of micronucleus and mutation induction by oxazepam in mammalian cells

The benzodiazepines are a class of drugs that are widely used in the treatment of various psychiatric disorders. One member of this class, oxazepam, is also a common metabolite of several other benzodiazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is inconsistent, we investigated the oxazepam-induced formation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and L5178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all three cell lines. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were formed in the first mitosis after treatment. Kinetochore staining (CREST-antiserum) revealed the presence of kinetochores in approximately 50% of the micronuclei in all three cell types. This result was further confirmed by in situ hybridization in L5178Y cells and indicates the presence of whole chromosomes or centric fragments as well as acentric fragments in the oxazepam-induced micronuclei. The L5178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used.     

A computerised kinetic database system for metallurgy and materials processing

An integrated and computerised kinetic database system, intellectualised database management system on kinetics of metallurgy (IDMSKM) is described in this report. IDMSKM has been designed using MS Windows and a Visual C⁺⁺ and Foxpro hybrid coding technique. It consists of two packages. The ThermoPhysData package has been established for physical property data retrieval and prediction, and presented in a previous publication in steel research. The emphasis in the present paper is placed on the KineticApp package. KineticApp involves G/S, S/S' and G/L' modules for the kinetic prediction, evaluation and analysis of gas/solid, solid/solid and gas/liquid reactions respectively. The key consideration in the module design is to establish the links of the kinetic model and parameters of a reaction system with the system characterisation. Assessed kinetic parameters stored in the database files in KineticApp include the activation energies for some reduction and decomposition reactions, as well as the diffusion in solid oxide systems. Those data are necessary while dealing with the reaction kinetic prediction problems. Several examples regarding the reduction of wustite pellet, the decomposition of CaCO₃ and the synthesis of SrTiO₃ are illustrated to show the features and functions of G/S and S/S modules.