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Ion-exchange properties of cell walls of Spinacia oleracea L. roots under different environmental salt conditions.

Ion-exchange properties of the polymeric matrix of cell walls isolated from roots of 55-day-old Spinacia oleracea L. (Matador cv.) plants grown in nutrient solution in the presence of 0.5, 150, and 250 mM NaCl and from roots of Suaeda altissima L. Pall plants of the same age grown in the presence of 0.5 and 250 mM NaCl were studied. The ion-exchange capacity of the spinach cell walls was determined at pH values from 2 to 12 and different ionic strength of the solution (10 and 250 mM NaCl). In the structure of the root cell walls, four types of ionogenic groups were found: amine, two types of carboxyl (the first being galacturonic acid residue), and phenolic groups. The content of each type of group and their ionization constants were evaluated. The ion-exchange properties of spinach and the halophyte Suaeda altissima L. Pall were compared, and the qualitative composition of the ion-exchange groups in the cell walls of roots of these plants appeared to be the same and not depend on conditions of the root nutrition. The content of carboxyl groups of polygalacturonic acid changed in the cell walls of the glycophyte and halophyte depending on the salt concentration in the medium. These changes in the composition of functional groups of the cell wall polymers seemed to be a response of these plants to salt and were more pronounced in the halophyte. A sharp increase in the NaCl concentration in the medium caused a decrease in pH in the extracellular water space as a result of exchange reactions between sodium ions entering from the external solution and protons of carboxyl groups of the cell walls. The findings are discussed from the standpoint of involvement of root cell walls of different plant species in response to salinity.

Cell Wall↗

Lysosomal elastase: effect on mechanical and biochemical properties of normal cartilage, inhibition by polysulfonated glycosaminoglycan, and binding to chondrocytes.

Rheumatic joint destruction usually starts with the destabilisation of cartilage. Lysosomal elastase is a candidate effector of this process, since this enzyme is found at the site of cartilage erosion by rheumatoid synovial tissue. In order to prove this hypothesis we assessed the mechanical stability of cartilage during treatment by this enzyme in vitro. An indentation apparatus was used for this purpose and biochemical as well as microscopic techniques were used to supplement the results thus obtained. Our findings show that elastase irreversibly impairs the stability of cartilage by lysis of matrix proteoglycans without the help of additive enzymes. Collagen fragmentation played no significant role during elastase-induced destabilisation, while specific collagenase attacked the collagen network within the matrix only subsequent to the removal of proteoglycans. These findings suggest that elastase is a leading enzyme during proteolytic cartilage degradation. In addition polysulfonated glycosaminoglycan was found to reduce the mechanical effect of elastase on normal cartilage. It is therefore concluded that local inhibition of elastase promises therapeutic benefit during rheumatic cartilage degradation. Upon treatment of cartilage with elastase we observed this enzyme not only within the matrix under destruction but also bound to chondrocytes. These findings support the hypothesis that elastase plays a role on the matrix not only by direct degradation, but also by an indirect effect mediated through living chondrocytes.

Adolescent↗

Framework for the environmental damage assessment of an industrial process chain.

On the basis of the life cycle approach, a framework for the assessment of the environmental damages generated by an industrial process chain is established. In order to consider all the processes of the life cycle, a methodology is developed based on an eco-matrix formed by chemical process eco-vectors containing all their environmental loads. To perform the impact assessment, environmental damage indicators are estimated in the most accurate way possible for each process. For this, site-specific evaluation is carried out applying damage functions and, hence, for each damage indicator, a damage matrix is obtained. In order to make the methodology more practical, options like dominance analysis are presented. Because of the large number of environmental loads, the focus is on the priority pollutants and then the final estimations are done with one indicator per safeguard subject. The damage indicators selected in this paper are damage costs, ecological damage parameter (both site-specific) and global damage estimates. As for the verification of the methodology, a study has been carried out based on the life cycle of the electricity produced by a municipal waste incinerator.

Animals↗

Matrix analysis for the dissection of interactions of G-protein beta3 subunit C825T genotype, allograft function, and posttransplant hypertension in kidney transplantation.

BACKGROUND: Complex relationships between genes and environment and the resulting biological impact have been dissected predominantly by conventional association studies. A major limitation of such studies results from the fact that only bidirectional investigations of genes and clinical end-points are commonly performed. The authors, therefore, applied matrix analyses to account for interactions between genetic and environmental factors influencing kidney allograft function. METHODS: By using matrices of correlation coefficients we tested the genetic effect of a variant within the gene encoding the beta3-subunit of heterotrimeric G-proteins (Gbeta3-C825T polymorphism) on posttransplant hypertension and kidney allograft function. This strategy allowed the authors to account for the influence of additional well-established genetic, clinical, and environmental confounders. The authors studied 281 consecutive white kidney recipients recruited between 1988 and 1993. Correlation coefficients of indices of relative change (percent) of systolic blood pressure (BP) and creatinine clearance (CrCl) were used in correlation coefficient matrices to elucidate interactions of parametrical biological parameters with environmental and genetic risk factors. RESULTS: A significant relationship was found between decreasing CrCl and increasing systolic BP in only those recipients who carried the Gbeta3-825TT genotype and did not lose graft during the first 3 years (R2 = 0.25; P = 0.021). CONCLUSIONS: In transplant recipients who did not lose their graft during the first 3 years after transplantation, the Gbeta3-TT genotype contributed to accelerated loss of allograft function by exaggeration of posttransplant hypertension. This relationship could only be elucidated by means of matrix analyses that allow the detection of complex relations between clinical, genetic, and environmental factors.

Adult↗

Simple and surprisingly effective one-step extraction-cleanup by soxflo for DDT and its metabolites from environmental samples.

A pilot study found that DDT breakdown at the GC inlet was extensive in extracts from some--but not all--samples with high organic carbon contents. However, DDT losses could be prevented with a one-step extraction-cleanup in the Soxflo instrument with dichloromethane and charcoal. This dry-column procedure took 1 h at room temperature. It was tested on spiked soil and peat samples and validated with certified soil and sediment reference materials. Spike recoveries from freshly spiked samples ranged from 79 to 111% at 20-4000 microg/kg concentrations. Recoveries from the real-world CRMs were 99.7-100.2% of DDT, 89.7-90.4% of DDD and 89.6-107.9% of DDE. It was concluded that charcoal cleanups should be used routinely during surveys for environmental DDX pollution in order to mitigate against unpredictable matrix-enhanced breakdown in the GC.

DDT↗

[Numerical identification of aquatic microorganisms using automated methods; for example, identification of bacteria from activated sludge].

A probability matrix for numerical identification of aquatic microorganisms was constructed using 90 miniaturized biochemical tests. More than 2000 reference strains originated from different culture collections and environmental strains identified by conventional methods were included in this matrix. All test result can be read visually and by photometer (automatically). First applications of this system on aerobic and facultative anaerobic heterotrophic organisms from activated sludge revealed the advantage of this system compared to conventional methods and delivered further a detailed description of the activated sludge community.

Bacteria↗

Decomposition of extremely hard-to-degrade animal proteins by thermophilic bacteria.

Hard-to-degrade animal proteins are ubiquitously present throughout animal bodies. Enormous numbers of these proteins generated in the meat industry are converted to industrial wastes, the disposal of which is tremendously difficult. Most hard-to-degrade animal proteins are currently disposed of by incineration; however, this method has ecological disadvantages in terms of an apparent energy loss and the production of a large amount of carbon dioxide. As a result, an innovative solution to these problems has been sought. In this review, we focus on the degradation of three hard-to-degrade animal proteins (extracellular matrix proteins, collagen in particular, keratin, and prion proteins) and discuss the decomposing capability of thermophilic bacteria. These proteins are strongly resistant to proteinases because of their structural features; therefore, new approaches employing bacterial proteases with strong activity and broad specificity are required for practical application.

Animals↗

Fully automated online solid phase extraction coupled directly to liquid chromatography-tandem mass spectrometry. Quantification of sulfonamide antibiotics, neutral and acidic pesticides at low concentrations in surface waters.

A fully automated online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) instrumental setup has been developed for the quantification of sulfonamide antibiotics and pesticides in natural water. The direct coupling of an online solid phase extraction cartridge (Oasis HLB) to LC-MS/MS was accomplished using column switching techniques. High sensitivity in the low ng/L range was achieved by large volume injections of 18 mL with a combination of a tri-directional auto-sampler and a dispenser system. This setup allowed high sample throughput with a minimum of investment costs. Special emphasis was placed on low cross contamination. The chosen approach is suitable for research as well as for monitoring applications. The flexible instrumental setup was successfully optimised for different important groups of bioactive chemicals resulting in three trace analytical methods for quantification of (i) sulfonamide antibiotics and their acetyl metabolites; (ii) neutral pesticides (triazines, phenylureas, amides, chloracetanilides) and (iii) acidic pesticides (phenoxyacetic acids and triketones). Absolute extraction recoveries from 85 to 112% were obtained for the different analytes. More than 500 samples could be analyzed with one extraction cartridge. The inter-day precision of the method was excellent indicated by relative standard deviations between 1 and 6%. High accuracy was achieved by the developed methods resulting in maximum deviation relative to the spiked amount of 8-15% for the different analytes. Detection limits for various environmental samples were between 0.5 and 5 ng/L. Matrix induced ion suppression was in general smaller than 25%. The performance of the online methods was demonstrated with measurements of concentration dynamics of sulfonamide antibiotics and pesticides concentrations in a little creek during rain fall events.

Anti-Bacterial Agents↗

Determination of fifteen priority phenolic compounds in environmental samples from Andalusia (Spain) by liquid chromatography-mass spectrometry.

This work describes the optimisation of a method to determinate fifteen phenolic compounds in waters, sediments and biota (green marine algae) by liquid chromatography coupled to mass spectrometry (LC-MS) with atmospheric pressure chemical ionisation (APCI) in the negative mode. The LC separations of the studied compounds and their MS parameters were optimised in order to improve selectivity and sensitivity. Separation was carried out with a C(18) column using methanol and 0.005% acid acetic as mobile phase in gradient mode. The molecular ion was selected for the quantitation in selective ion monitoring (SIM) mode. A solid-phase extraction (SPE) method was applied in order to preconcentrate the target analytes from water samples. However, extraction of the compounds from sediment and biota samples was carried out by liquid-solid extraction with methanol/water after studying the influence of other organic solvents. In addition, a clean-up step by SPE with HLB Oasis cartridges was necessary for sediments and biota. The proposed analytical methodology was validated in the target environmental matrices by the analysis of spiked blank matrix samples. Detection limits were 10-50 ng L(-1) for water, 1-5 microg kg(-1) for sediments and 2.5-5 microg kg(-1) for biota samples. Good recoveries and precision values were obtained for all matrices. This methodology has been successfully applied to the analysis of incurred water, sediment and biota samples from Andalusia (Spain).

Biodiversity↗

The protein composition of normal and developmentally defective enamel.

The development of human enamel involves a complex series of events including the secretion and degradation of a unique extracellular matrix. Ameloblasts progress through a succession of cellular phenotypes executing specialized secretory and regulatory functions. When performing optimally, ameloblasts produce a highly structured and mineralized tissue. Given the elaborate developmental events required for normal enamel formation, it is not surprising that a variety of enamel malformations arise from defects in matrix synthesis, secretion and extracellular processing. Normal matrix secretion and post-secretory processing by ameloblasts can be affected by a variety of hereditary and environmental conditions. These disturbances can result in an abnormal amount and/or composition of matrix proteins, and subsequently, an altered enamel structure and/or mineral content. For example, abnormal matrix removal during enamel maturation apparently contributes to hypomineralization associated with dental fluorosis. Incomplete matrix removal can also occur in several different forms of the hereditary condition amelogenesis imperfects. Specific types of this condition can have retention of substantial enamel protein (e.g. 5% by weight) that is, at least in part, composed of amelogenin and/or its breakdown products. Characterization of the enamel proteins in teeth affected by developmental disturbances can provide insight into the pathogenesis and normal formation of this highly specialized tissue.

Adult↗

Clinical biophysics: the promotion of skeletal repair by physical forces.

Skeletal tissues respond to the physical demands of their environment by altering the synthesis and organization of the extracellular matrix. These observations have major implications for how physical environmental demands result in the clinical observations of atrophy and hypertrophy, and how manipulation of the physical environment can be used therapeutically to stimulate repair. Electrical stimulation will be considered as a paradigm of how musculoskeletal tissues respond to physical stimuli. A model of demineralized bone matrix-induced endochondral ossification has been used because it epitomizes the cell biology of endochondral bone formation in a temporally consistent way. We have studied cartilage and bone matrix production, the temporal locus of cell responsiveness, signal dosimetry, and the synthesis of signaling cytokines (TGF-beta) using biochemical, immunohistochemical, and molecular techniques. Exposure to certain electrical environments enhances chondrocyte differentiation reflected as a temporal acceleration and quantitative increase of cartilage extracellular matrix, earlier onset of osteogenesis, and more mature trabecular bone. The cell pool competent to respond resides in the mesenchymal stage. The enhancement in chondrogenesis is associated with an increase in TGF-beta synthesis mediated at least in part by binding of the transcription factor AP-1 and may be modulated specifically by phosphorylation of JNK. The clinical practice of orthopedics has empirically created a variety of biophysical environments in attempts to optimize skeletal repair. We are beginning to understand the biological effects of biophysical stimulation and are now poised to replace empiricism with treatment paradigms based upon physiologic understandings of dose and biologic response.

Animals↗

Modulation of glial glutamate transport through cell interactions with the extracellular matrix.

Glial glutamate transport plays a pivotal role in maintaining glutamate homeostasis in the central nervous system. Expression of glutamate transporters is highly regulated during brain development, and a number of pathological conditions are associated with deficits in expression and/or function of glutamate transports. While several soluble factors have been shown to regulate the expression of glutamate transporter, the contribution of cell-cell interaction and cell-environmental interaction in the regulation of glutamate transport is unknown. Extracellular matrix (ECM) molecules are essential components in cell-cell and cell-environmental interactions, and the ECM has been shown to play critical role in normal development and during brain pathogenesis. We, therefore, investigated the possibility that ECM molecules may regulate astrocytic glutamate transport. Therefore, we cultured rat cortical astrocytes with different ECMs and determined expression levels of the two astrocytic glutamate transporters GLT-1 and GLAST by Western Blot and determined transporter activity through measurements of 3H-D-aspartate uptake. Astrocytes grown on poly-ornithine or poly-D/L-lysine showed approximately two-fold higher GLT-1 expression than sister cells grown on plastic dishes without ECM. Naturally occurring ECM's, including laminin and collagen, showed a dose-dependent regulation of GLT-1 protein expression. These effects were specific for GLT-1 as GLAST expression was unaffected by different ECMs. Surprisingly, however, none of the examined ECMs altered the apparent glutamate uptake activity. In probing blots side-by-side for expression of Na(+)/K(+)-ATPase, we found that ECMs affected expression of Na(+)/K(+)-ATPase and GLT-1 in a reciprocal fashion. Poly-ornithine, for example, enhanced GLT-1 expression, but reduced expression of Na(+)/K(+)-ATPase. Na(+) transport may, thus, be a limiting factor for glutamate uptake.

Amino Acid Transport System X-AG↗

Sugar additives for MALDI matrices improve signal allowing the smallest nucleotide change (A:T) in a DNA sequence to be resolved.

Sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of DNA is critical for obtaining high quality mass spectra. Sample impurity, solvent content, substrate surface and environmental conditions (temperature and humidity) all affect the rate of matrix-analyte co-crystallization. As a result, laser fluence threshold for desorption/ionization varies from spot to spot. When using 3-hydroxypicolinic acid (3-HPA) as the matrix, laser fluence higher than the threshold value reduces mass resolution in time-of-flight (TOF) MS as the excess energy transferred to DNA causes metastable decay. This can be overcome by either searching for 'hot' spots or adjusting the laser fluence. However, both solutions may require a significant amount of operator manipulation and are not ideal for automatic measurements. We have added various sugars for crystallization with the matrix to minimize the transfer of excess laser energy to DNA molecules. Fructose and fucose were found to be the most effective matrix additives. Using these additives, mass resolution for DNA molecules does not show noticeable deterioration as laser energy increases. Improved sample preparation is important for the detection of single nucleotide polymorphisms (SNPs) using primer extension with a single nucleotide. During automatic data acquisition it is difficult to routinely detect heterozygous A/T mutations, which requires resolving a mass difference of 9 Da, unless a sugar is added during crystallization.

DNA Mutational Analysis↗

Interplay between epidermal growth factor receptor and Janus kinase 3 regulates polychlorinated biphenyl-induced matrix metalloproteinase-3 expression and transendothelial migration of tumor cells.

We hypothesize that environmental toxicants, such as polychlorinated biphenyl congeners, can activate vascular endothelial cells and thus increase formation of blood-borne metastases. This study indicates that exposure of human microvascular endothelial cells to 2,2',4,6,6'-pentachlorobiphenyl can stimulate transendothelial migration of tumor cells through up-regulation of matrix metalloproteinase (MMP)-3. In a series of experiments with specific small interfering RNA and pharmacologic inhibitors, we provide evidence that 2,2',4,6,6'-pentachlorobiphenyl can activate epidermal growth factor receptor (EGFR) and Janus kinase 3 (JAK3) in a closely coordinated and cross-dependent fashion. Activated EGFR and JAK3 stimulate in concert c-Jun NH(2)-terminal kinase and extracellular signal-regulated kinase 1/2 as well as increase DNA-binding activity of transcription factors activator protein-1 and polyomavirus enhancer activator protein 3, leading to transcriptional up-regulation of MMP-3 expression. These results indicate that the interplay among EGFR, JAK3, and mitogen-activated protein kinases, such as c-Jun NH(2)-terminal kinase and extracellular signal-regulated kinase 1/2, is critical for polychlorinated biphenyl-induced MMP-3 expression and accelerated transendothelial migration of tumor cells.

Breast Neoplasms↗

A stochastic mechanism for biofilm formation by Mycoplasma pulmonis.

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.

Antigens, Bacterial↗

Proliferation and collagen synthesis of human anterior cruciate ligament cells in vitro: effects of ascorbate-2-phosphate, dexamethasone and oxygen tension.

Clinical and experimental studies demonstrate that injured anterior cruciate ligaments (ACL) do not usually heal and that autografts used to repair the ACL rapidly weaken in the early period and take a long time to regain strength. The aim of this study was to develop an in vitro culture system in which environmental and biochemical factors influencing the proliferation and matrix synthesis of cells derived from human anterior cruciate ligaments can be studied. Primary cultures of human ACL cells were obtained by outgrowth from explants of normal ACL obtained at knee replacement for osteoarthritis in Dulbecco's minimum essential medium (DMEM). The effects of the additives 100 microm L-ascorbic acid-2-phosphate (Asc-2-P) and 10 n m dexamethasone (dex) on proliferation and collagen synthesis were assessed after 4, 8 and 12 days in culture. Ligament cells were grown at 0, 5, 10 and 21%p O(2)in the presence of 100 microm asc-2-P and 10 n m dex. DNA content was assessed using the Hoechst dye method and collagen synthesis by the incorporation of 5 mCi/ml [(3)H]proline after 3, 6 and 12 days in culture. At 21%p O(2), the presence of asc-2-P and dex induced significantly greater (P< 0.01, ANOVA) cell proliferation than with either additives alone. Greatest percentage collagen to total protein synthesis was observed when cells were grown in the presence of asc-2-P only. Least proliferation and percentage collagen to total protein synthesis was seen when both additives were omitted. Greatest cell proliferation was seen when cells were grown in 10%p O(2)and 5%p O(2)was associated with increased collagen synthesis. These results suggest that it is possible to study the effects of environmental and biochemical factors on human ACL healing in vitro. Our data suggest oxygen can influence certain biosynthetic activities of ACL cells. Low oxygen tensions lead to an increase in collagen production by ACL cells. However early responses to injury require extensive cell proliferation which may be activated at higher p O(2). Variation of p O(2)in ligaments during healing may therefore be an important modulator of successful repair.

Anterior Cruciate Ligament↗

Fluorescence characterization of the interaction of Al3+ and Pd2+ with Suwannee River fulvic acid in the absence and presence of the herbicide 2,4-dichlorophenoxyacetic acid.

In an effort to understand the role of environmental metal ions in the interaction of charged pesticides with humic substances, a fluorescence study of the interaction of the widely-used herbicide 2,4-dichlorophenoxyacetic acid (DCPAA) with Al(3+) and Pd(2+) and Suwannee River fulvic acid (SRFA) was undertaken. Initial fluorescence experiments on binary solutions clearly indicated that both Al(3+) and Pd(2+) strongly interact with both SRFA and DCPAA when alone in solution with the metal ion. Titrations of SRFA with Al(3+) at pH values of 4.0, 3.0 and 2.0 revealed decreased degrees of fluorescence emission enhancement (at lambda(emission, max)=424 nm) with decreasing pH, consistent with the expected loss of rigidity in the SRFA-Al(3+) complexes formed as pH is lowered. In contrast, titrations of SRFA with Pd(2+) at all of these pH values resulted in significant fluorescence quenching. Al(3+) additions to solutions of DCPAA at pH values above the pK(a) (2.64) of DCPAA resulted primarily in significant changes in the wavelength of maximum emission (without significant quenching or enhancement of emission intensity), while Pd(2+) additions to DCPAA solutions resulted primarily in very significant fluorescence quenching. The DCPAA fluorescence results strongly support the formation of an Al(3+)-DCPAA complex at pH values above the pK(a) of DCPAA. The fluorescence results obtained for solutions of Pd(2+) and DCPAA are best explained by a collisional quenching mechanism, that is, energy transfer from excited DCPAA molecules to Pd(2+) following the collision of these two species in solution. Excitation-emission matrix plots obtained on ternary solutions (at environmentally-relevant pH 4.0) containing SRFA, DCPAA and metal ions (i.e., either Al(3+) or Pd(2+)) provides evidence (especially for systems containing Al(3+)) for the existence of ternary complexes between fulvic acid species, the herbicide DCPAA and metal ion, suggesting (at least at pH 4.0, where the predominant DCPAA species is negatively-charged) that metal ions may function to "bridge" negatively-charged fulvic acids to negatively-charged pesticides.

2,4-Dichlorophenoxyacetic Acid↗

Biofilm formation by a Bacillus subtilis strain that produces gamma-polyglutamate.

The extracellular matrix produced by Bacillus subtilis B-1, an environmental strain that forms robust floating biofilms, was purified, and determined to be composed predominantly of gamma-polyglutamate (gamma-PGA), with a molecular mass of over 1,000 kDa. Both biofilm formation and gamma-PGA production by B. subtilis B-1 increased with increasing Mn(2+) or glycerol concentration. gamma-PGA was produced in a growth-associated manner in standing culture, and floating biofilms were formed. However, gamma-PGA was produced in a non-growth-associated manner in shaking culture conditions. When B. subtilis B-1 was grown in a microaerated culture system, floating biofilm formation and gamma-PGA production were significantly retarded, suggesting that oxygen depletion is involved in the initial steps of floating biofilm formation in standing culture. Proteomic analysis of membrane proteins demonstrated that flagellin, oligopeptide permease and Vpr protease precursor were the major proteins produced by cells in a floating biofilm and a colony.

Bacillus subtilis↗