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PubMedDate not supplied

Chemical and immunological characterization of oxidative nonenzymatic protein modifications in dialysis fluids.

BACKGROUND: Glucose degradation products (GDP) in dialysis fluids may induce nonenzymatic protein modifications, the chemical nature and biological properties of which should be better defined. AIMS: To characterize nonenzymatic protein modifications present in glucose-based peritoneal dialysis fluids (PDF) and to evaluate the relationship between concentrations of GDP and the derived nonenzymatic modifications, and the potential of PDF for generating these modifications in vitro. METHODS: The presence, distribution, and content of several nonenzymatic protein modifications in PDF were evaluated by immunological methods, by HPLC, and by gas chromatography-mass spectrometry (GC/MS). Peritoneal dialysis fluid-induced oxidative stress in cells was evaluated by flow cytometry. The potential of PDF for generating oxidative and glycoxidative modifications was examined by immunological and cross-linking analyses. RESULTS: The albumin present in PDF is modified by carboxymethyllysine (CML). GC/MS analyses of PDF proteins confirmed the presence of CML and demonstrated the occurrence of carboxyethyllysine, malondialdehyde lysine, and oxidation-derived semialdehydes. Furthermore, their concentrations in PDF proteins were significantly higher than those in plasma proteins (in all cases, p < 0.02). The concentration of pyrraline, a non-oxidative advanced glycation end-product, increased with dwell time up to 6 hours (p < 0.03). The PDF induced cellular free-radical production, which was partially inhibited by the Maillard reaction inhibitor aminoguanidine (p < 0.001). The potential to generate oxidative and glycoxidative modifications demonstrated an inverse relationship with dwell time (p < 0.05). The PDF was able to induce collagen cross-linking in a close relationship with GDP concentration. CONCLUSIONS: (1) PDF contains non-oxidative and several oxidative nonenzymatic protein modifications in higher concentrations than plasma. (2) Peritoneal dialysis fluid induces oxidative stress in vitro, which can be partially inhibited by aminoguanidine. (3) These properties are directly related to GDP concentration. (4) Peritoneal dialysis fluid is able to generate glycoxidative and oxidative damage to proteins in vitro in a dwell-time dependent fashion.

Dialysis Solutions↗

PubMed1980

Virus adaptation to host cells: the non-classical modification of phage T3.

Bacterial virus T3 undergoes host-controlled modification which is not based on "classical" processes of DNA modification and restriction. The adsorption and thus the growth of T3 on Escherichia coli W cells (E. coli K12 derivative) decisively depends on the host strain on which the virus was previously propagated. Depending on the modification conferred to the virus by its last host, its efficiency of plating (e.o.p.) on E. coli W varies by six orders of magnitude between 10(-7) and 10(-1). This does not reflect the appearance of T3 host-range mutants, but a fully reversible modification of genotypically unchanged T3 wild-type phage. The behaviour of T3 in the described host system constitutes a second case of so-called non-classical modification and restriction (KRUGER et al. 1977, Molec. gen. Genet. 153, 107-110) of bacteriophages. Non-classical modification (protein modification) is additive to and independent of DNA modification and restriction as demonstrated with the ocr- phage T3/R7. - Furthermore, our results suggest that the adsorption specificity of T3 is determined by at least two independent genetic factors; in both of these factors T3 differs from T7.

Adaptation, Physiological↗

PubMed1995

Protein thiol modification of glyceraldehyde-3-phosphate dehydrogenase as a target for nitric oxide signaling.

Nitric oxide signaling is achieved through cGMP-dependent and -independent mechanisms. The latter are exemplified by the NAD(+)-dependent automodification of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The experimental post-translational, covalent modification of the enzyme by [32P]NAD+ is achieved using NO-releasing compounds and an active constitutive or inducible NO-synthase. Potential roles for NO in this covalent enzyme modification can be grouped as follows: S-Nitrosylation of GAPDH by NO+ NAD(+)-dependent, post-translational covalent automodification of GAPDH. Oxidative modification of GAPDH by NO-related compounds, probably ONOO. GAPDH modification by one of the proposed mechanisms would lead to inhibition of enzyme catalysis. It is likely that the NAD(+)-dependent automodification process occurs in vitro, in intact cells, and in whole animals. Besides its normal function in glycolysis, GAPDH not only is a target for NO-mediated direct and indirect modifications but also is ADP-ribosylated in the presence of brefeldin A (90). The relation of such ADP-ribosylation to enzyme activity is so far unknown. GAPDH also may be involved in one of the following functions unrelated to its glycolytic activity (81 and refs. therein; 90): binding and transport of tRNA associated with nuclear localization of GAPDH. DNA-repair activity, i.e., uracil DNA glycosylase. Activation of transcription in neurons. Interaction with tubulin and microtubules. The transport of nitric oxide. Serves as a substrate for brefeldin A stimulated ADP-ribosylation. Because some of these alternative functions of GAPDH, just like NO-mediated modification of the enzyme, are related to the NAD+ binding site of the protein, we are interested in searching for the significance of these activities in relation to NO actions. In recent years, several functions of NO have been linked to direct, cGMP-independent actions. Modification of GAPDH is probably just one interesting target related to NO-redox chemistry and active-site thiol modification. It will be challenging to investigate NO biochemistry in closer detail and to elucidate how NO targets biological systems, especially in relation to the patho-physiological role of NO in medically related conditions.

Amino Acid Oxidoreductases↗

PubMed1993

Ascorbic acid oxidation product(s) protect human low density lipoprotein against atherogenic modification. Anti- rather than prooxidant activity of vitamin C in the presence of transition metal ions.

The oxidative modification of low density lipoprotein (LDL) has been proposed as an important causative event in the development of human atherosclerosis. As a corollary of this hypothesis, antioxidants that can prevent LDL oxidation may inhibit atherosclerosis. Oxidative modification of LDL in vitro, either induced by Cu2+ or mediated by cultured arterial wall cells in media containing trace amounts of transition metal ions, is strongly inhibited by vitamin C (L-ascorbic acid (AA)). AA, however, is known to act as a prooxidant rather than an antioxidant in the presence of transition metal ions. We observed that AA is oxidized rapidly when incubated with Cu2+ and LDL, leading to transient formation of dehydro-L-ascorbic acid (DHA). Although AA and DHA can no longer be detected after 3.5 h of incubation, LDL resists oxidative modification for at least 20 h, as assessed by anodic gel electrophoretic mobility. Remarkably, DHA protects LDL more effectively against both Cu(2+)-induced lipid peroxidation and shifts in electrophoretic mobility than does AA; indeed, AA per se, without oxidation to DHA, offers no protection. By inhibiting oxidative modification of LDL, AA and DHA prevent uptake of LDL by macrophages via the scavenger receptor pathway. When LDL is incubated with DHA followed by gel filtration, LDL remains protected against subsequent Cu(2+)-induced oxidative modification, suggestive of stable modification of LDL in the presence of DHA. In contrast, DHA is ineffective against a metal ion-independent type of oxidative stress, viz. aqueous peroxyl radicals; under these conditions, only AA is able to inhibit lipid peroxidation in LDL. Our data indicate that vitamin C protects LDL against atherogenic modification by two different mechanisms that may act in concert: (i) free radical scavenging by AA prevents aqueous oxidants from attacking and oxidizing LDL, and (ii) stable modification of LDL by DHA or decomposition product(s) thereof imparts increased resistance to metal ion-dependent oxidation.

Adult↗

PubMedDate not supplied

Modification of human LDL by in vitro incubation with cigarette smoke or copper ions: implications for allergies, asthma and atherosclerosis.

Cigarette smoking has been linked to a higher risk of not only atherosclerosis and related diseases, but asthma and allergies as well. The mechanisms linking smoking to these diseases may be due in part to increased low density lipoprotein (LDL) modification. In the current study, we compared the modification in vitro of LDL isolated from healthy volunteers that had been exposed to either the gas phase of cigarette smoke or copper ion mediated oxidation. The study used as measures of modification/damage the levels of protein carbonyl groups, changes in electrophoretic mobility on agarose gel electrophoresis and levels of thiobarbituric-reacting substances. Other measures used to assess other aspects of LDL modification included SDS PAGE and immunoblotting. Both copper ion or exposure to the gas phase of cigarette smoke increased electrophoretic mobility of LDL but the increase was greater in the gas phase smoke group. In contrast, thiobarbituric reacting substance levels were increased primarily in copper oxidized LDL. Protein carbonyl levels were increased to a similar extent in both copper ion and smoke exposed samples. Addition of EDTA prevented the modifications found upon copper mediated oxidation of LDL, but EDTA did not prevent the modification of the gas phase cigarette smoke exposed LDL. In summary, the results indicate that protein carbonyl formation can be used as a measure of the modification of LDL particles and, using several different assessment techniques, there are distinct differences in the modified LDL produced by in vitro incubation with gas phase cigarette smoke relative to that found upon incubation of LDL with copper ion. The in vitro smoking-produced LDL modifications may potentially be relevant to the process of lipoprotein modification in vivo and to the subsequent biological effects of these modified lipoproteins on processes affected by the immune system involvement, such as atherosclerosis and allergy/asthma.

Aerosols↗

PubMed1996

Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding.

Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules. Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding. This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt. A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions. Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding. A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E. coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP). Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E. coli RNase P RNA. Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes.

Animals↗

PubMed1998

Modification of tau to an Alzheimer's type protein interferes with its interaction with microtubules.

The microtubule associated protein tau is the main structural component of paired helical filaments (PHFs), aberrant polymers found intracellularly in neurons of brains with the Alzheimer's disease. Glycation is one of the posttranslational modifications that has been found in tau from PHFs, but not in normal brain tau. Studies were carried out with purified tau protein subjected to chemical modifications, in order to further investigate the mechanisms of tau self-association into PHFs. Tau was subjected to modifications affecting reactive lysyl residues, e.g., carbamoylation with potassium cyanate and glycation reaction with glucose. The effects of these modifications to produce functional alterations in tau capacity to bind brain tubulin and to induce microtubule assembly were investigated. Chemically-modified tau and tau of Alzheimer's type exhibited a similar microtubule interaction behavior as analysed by overlay assays, but those were different than normal tau controls. On the other hand, studies of the microtubule assembly kinetics indicated that the reported tau modifications resulted in a loss of its capacity to promote microtubule assembly from purified tubulin preparations. The data on the differences in the electrophoretic profiles, Western blots and the overlay patterns, along with those on the microtubule polymerisation of normal brain tau as compared with both modified and Alzheimer's tau, suggest changes in the functional behavior of this protein as a result of its structural modifications. These studies were complemented with an immunogold analysis at the electron microscope level, which indicated that the modified tau did not incorporate into assembled microtubules. These findings, combined with the results on tau chemical modifications suggest that the reactive lysine residues within functional domains on tau, e.g., those of the repetitive binding motifs, were affected by these modifications. Furthermore, these observations provide new clues to understand the anomalous interactions of tau in Alzheimer's disease.

Alzheimer Disease↗

PubMed2003

Restriction-modification system in bacteriophage MB78.

Restriction-modification system is present in bacteria to protect the cells against phage infection. Interestingly, the bacteriophage MB78, a virulent phage of Salmonella typhimurium possesses restriction-modification system. Permissive host transformed with plasmid having the genomic fragment of MB78 carrying the putative restriction-modification genes severely restrict the growth of the phage 9NA. Growth of phage MB78 is also restricted to some extent. However, the temperate phage P22 is not restricted at all. Cloning of the the putative restriction-modification genes has been done in both orientations in different vectors. The clones carrying the genes in the same orientation as that of the lacZ in pUC19 are mostly unstable. However, those are stable when cloned in opposite orientation. Viability of the transformants is strain-, orientation-, and medium-dependent. The two genes have also been cloned individually/separately. Hosts carrying only the modification gene do not restrict growth of phages while the hosts carrying only the restriction gene do. The former produces stable transformants while the latter produces very unstable transformants which were viable only upto 36 h or so. The colonies carrying modification gene were normal looking while those carrying the restriction gene were tiny, flat, and looked distressed resembling very much the clones carrying bacterial restriction-modification system. Amplification of the genes and subsequent cloning in expression vector will be carried out for characterization of the enzymes.

Base Sequence↗

PubMed2026

m6A RNA modification and its emerging roles in diseases: recent advances and therapeutic implications.

BACKGROUND: In the recent past, insights in post transcriptional regulation of gene expression have profoundly reshaped our understanding of the molecular mechanisms underlying health and disease. This paradigm shift largely stems from the emerging field of epitranscriptomics, which highlights the pivotal role of chemical RNA modifications. While more than 170 distinct chemical modifications on the RNA are known, the m6A modification is the most abundant internal mRNA modification in higher eukaryotic cells, present not only on protein coding transcripts but also on non-coding RNAs, regulated by “writers”, “erasers”, and “readers” that together modulate alternative splicing, nuclear export, translation efficiency, and mRNA stability. MAIN BODY: This review addresses an important gap by presenting a multilayered regulatory framework that catalogs the full repertoire of m6A machinery and uniquely reveals how non-coding RNAs, transcription factors, histone modifications, and chromatin remodelers governs the spatiotemporal specificity of m6A modification. We explore how dysregulation of m6A modification and its regulatory proteins contribute to the development and progression of various diseases such as cardiovascular disease, neurological disorders, cancer, and type 2 diabetes through context-dependent modulation of gene networks. Furthermore, we present an integrative overview of the therapeutic pipeline, tracing the development of small-molecule inhibitors targeting m6A regulators, thus bridging a crucial link between fundamental mechanisms and new therapies. CONCLUSIONS: Overall, this review integrates current findings and emerging insights to provide a comprehensive understanding of m6A biology. By linking upstream regulatory mechanisms with downstream pathological consequences and therapeutic interventions, we highlight the potential of targeting the epitranscriptome for clinical applications.

Humans↗

PubMed2005

Post-translational modifications influence transcription factor activity: a view from the ETS superfamily.

Transcription factors provide nodes of information integration by serving as nuclear effectors of multiple signaling cascades, and thus elaborate layers of regulation, often involving post-translational modifications, modulating and coordinate activities. Such modifications can rapidly and reversibly regulate virtually all transcription factor functions, including subcellular localization, stability, interactions with cofactors, other post-translational modifications and transcriptional activities. Aside from analyses of the effects of serine/threonine phosphorylation, studies on post-translational modifications of transcription factors are only in the initial stages. In particular, the regulatory possibilities afforded by combinatorial usage of and competition between distinct modifications on an individual protein are immense, and with respect to large families of closely related transcription factors, offer the potential of conferring critical specificity. Here we will review the post-translational modifications known to regulate ETS transcriptional effectors and will discuss specific examples of how such modifications influence their activities to highlight emerging paradigms in transcriptional regulation.

Animals↗

PubMed2003

Wavelength and average power density dependency of the surface modification of root dentin using an MIR-FEL.

BACKGROUND AND OBJECTIVES: Surface modification of root dentin by mid-infrared (MIR) pulsed-laser irradiation is one of the candidates for a novel, non-invasive treatment to prevent root surface caries. To modify root dentin effectively and non-invasively it is essential to estimate quantitatively and qualitatively the laser parameters, such as the wavelength and power density, required for surface modification. The key aspect is to bring about effective surface modification of the root dentin while minimizing the unwanted removal of the underlying dentin. STUDY DESIGN/MATERIALS AND METHODS: Using a tunable, MIR Free Electron Laser with lambda = 8.8-10.6 microm, we have investigated macroscopically the extent of the surface modification (morphological and chemical changes) of root dentin. We have obtained experimental results related to the ablation depth, the MIR absorption spectrum, and the elemental chemical composition. RESULTS: The observations showed that the surface modification of root dentin was inclined toward well-recrystallized HAp-like material, leading to an increase in the acid resistance and dentinal tubule sealing. The laser parameters, at which efficient surface modification without enhanced ablation occurred, were estimated to be approximately in the wavelength region around lambda = approximately 9.0 or approximately 9.7 microm and in the average power density region of approximately 10-20 W/cm2 (resulting in total energy density and peak power density regions of approximately 1-2 kJ/cm2 and approximately 0.67-1.2 kW/cm2). CONCLUSIONS: The surface modification of root dentin strongly depends on the laser parameters applied. We conclude that the optimum wavelengths for laser treatment of root surface caries are lambda = approximately 9.0 or approximately 9.7 microm, corresponding to the absorption peak due to P-O stretching.

Animals↗

PubMed1993

Retinoids inhibit the oxidative modification of protein kinase C induced by oxidant tumor promoters.

Recently we reported that oxidant tumor promoters can induce the oxidative modification of protein kinase C (PKC) resulting in either activation or inactivation of the kinase (R. Gopalakrishna and W. B. Anderson, Arch. Biochem. Biophys. 285, 382-387, 1991). Since retinoids previously have been shown to antagonize the actions of tumor promoters, studies were carried out to determine if retinoids can inhibit the oxidative modification of PKC induced by tumor promoters. Prior treatment of B16 melanoma cells or C6 glioma cells with all-trans-retinoic acid (0.1 microM) for a short time period (15 to 60 min) followed by subsequent treatment with oxidants such as hydrogen peroxide resulted in a 30 to 70% decrease in the oxidative modification of PKC. This resulted in a decrease in oxidant-induced conversion of PKC from a Ca2+/lipid-dependent form (peak A) to a Ca2+/lipid-independent form (peak B). This retinoid-mediated protection also was observed with the reversible oxidative modification of PKC induced by m-periodate treatment of intact cells. To understand whether this protection offered by retinoids was caused by a direct influence of retinoids on PKC, experiments were carried out using the purified enzyme. The results of experiments using isolated PKC suggested that retinoids can act directly to protect the regulatory domain of PKC from oxidative modification induced by oxidants. However, high (1-10 microM) concentrations of retinoids are necessary to elicit this protection of isolated PKC. In contrast, in experiments with intact cells, only low (submicromolar) concentrations of retinoids are required to protect PKC from oxidation. The differences noted in the retinoid concentrations required to protect PKC from oxidant modification in the test tube versus in the intact cell may be due to increased retention of retinoids in the cell membrane by partitioning, or to other indirect actions of retinoids in the intact cells to decrease cellular oxidations. These results suggest that some of the anti-tumor promoter actions of retinoids may be mediated, in part, by inhibiting the oxidative modification of protein kinase C induced by oxidant tumor promoters.

Animals↗

PubMed1995

Effect of diabetes and dietary ubiquinone supplementation on the post-translational modification of rat lens beta L crystallin.

The effect of streptozocin diabetes of 14 days duration on the integrity of lenticular crystallins has been determined by the measurement of characteristic markers of protein modification in the lens crystallins of rats. Further, the susceptibility of the crystallins to modification has also been determined by measurement of the same markers after the application of a metal-catalyzed oxidative insult in vitro. The results show that the previously reported increased post-translational modification of lens crystallins in vivo and increased susceptibility to modification in vitro of diabetic crystallins after 21 days of uncontrolled diabetes are also evident after just 14 days of diabetes. Treatment of the diabetic animals with the antioxidant ubiquinone by dietary supplementation was unable to prevent the post-translational modifications sustained by the crystallin when subjected to diabetes in vivo or the increase in susceptibility to an in vitro oxidative stress. While the present results support the proposal that cataract formation is initiated by protein post-translational modification factors such as glycation, ubiquinone supplementation does not appear to be beneficial in the inhibition of post-translational crystallin modification in diabetic cataractogenesis.

Animals↗

PubMed1990

Chemical modification studies on a blood group A-specific lectin, crotalarin (Crotalaria striata) and its effect on hemagglutinating activity.

Crotalarin, the N-acetyl-D-galactosamine-binding blood group A-specific lectin from the seeds of Crotalaria striata was subjected to various chemical modifications in order to ascertain the amino acid residues responsible for its carbohydrate-binding property. Modification of lysine, cysteine and arginine residues did not affect the carbohydrate-binding activity of the lectin. However, modification of tyrosine residue and carboxy group of the acidic amino acids led to a complete loss of its activity, indicating the involvement of tyrosine and aspartic and glutamic acid in the saccharide-binding respectively. The hemagglutinating activity of the lectin was completely/almost completely lost by modification of tryptophan residues. The relative loss in hemagglutinating activity on modification of tryptophan residues indicate that one residue/molecule is required for the carbohydrate-binding activity of the lectin. Modification was not effective in the presence of D-galactose (0.2 M). A marked decrease in the fluorescence emission was found as the tryptophan residues of crotalarin were modified. The c.d. spectra showed the presence of an identical pattern of conformation in the native and modified lectins which confirms that the loss in activity was due to modification only. The effect of periodate oxidation on crotalarin showed loss of activity whereas action of enzymes retained most of the activity.

ABO Blood-Group System↗

PubMed1984

Amino group modification of (Na+ + K+)-ATPase.

The effects of three amino group reagents on the activity of (Na+ + K+)-ATPase and its component K+-stimulated p-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na+ of Mg2+ present, the enzyme inactivation process follows complicated kinetics. In the presence of K+, Rb+, or Tl+, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg2+ (P50 6 microM vs. 1.2 mM). Under certain conditions (100 microM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na+ + K+)-ATPase. Mg2+ and K+ have no effects, and ATP only a minor effect, on the degree of modification. The K+-stimulated p-nitrophenylphosphatase activity is less inhibited than the (Na+ + K+)-ATPase activity. Half-inhibition of the (Na+ + K+)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na+ + K+)-ATPase. Mg2+ has no effect, and ATP has only a slight protecting effect. The K+-stimulated p-nitrophenylphosphatase activity is inhibited in parallel with the (Na+ + K+)-ATPase activity. Half-inactivation of the (Na+ + K+)-ATPase activity is obtained after 20% modification of the amino groups.

4-Nitrophenylphosphatase↗

PubMed1998

Influence of chemical modification of cysteine and histidine side chains upon subunit reassembly of alpha crystallin.

Alpha crystallin, the important multimeric structural protein of mammalian eye lens, is an assembly composed of 30 alpha-A and 10 alpha-B subunits. The influence of either partial or complete chemical modification of two important amino acid side chains, cysteine and histidine, upon the integrity of native alpha crystallin assembly and also upon the mode of subunit reassembly has been investigated. It has been found that chemical modification of surface-exposed cysteine and histidine side chains does not affect the subunit-subunit interactions stabilizing the native aggregate. Cysteine modifications, either partial or complete, unlike histidine modifications, do not seem to affect the backbone conformation of the subunits refolded after denaturation. Both cysteine and histidine modifications, however, affect the packing of the refolded structural elements forming the tertiary structure of the subunits and also the mode of oligomeric reorganization. The most striking effect of histidine modification is the considerable increase in size of the aggregates upon reassociation of the modified subunits. The chaperone activity, however, has been found to remain almost unaffected in spite of these chemical modifications.

Chromatography, Gel↗

PubMed1998

An acidic modification of the cytoplasmic domain contributes to the charge heterogeneity of the MHC class I antigens.

Polypeptide phosphorylation and sialylation of the glycan moieties contribute to the charge heterogeneity of the class I major histocompatibility complex glycoproteins. The present study demonstrates that a unique acidic modification unrelated to phosphorylation or glycosylation also affects the charge heterogeneity of the H2-Kk heavy chain of BW5147 lymphoma cells. In vitro cultivation of BW5147 cells results in changes in charge heterogeneity of the H2-Kk heavy chains due to the unique acidic modification. Sequential papain digestion of the 45 000 Mr H2-Kk glycoprotein yields a 42 500 Mr glycopolypeptide initially, followed by production of a 39 000 Mr glycopolypeptide. Results from experiments designed to localize and characterize the novel acidic modification suggest that the modification resides in the segment of the H2-Kk polypeptide located between the two papain cleavage sites. This portion of the polypeptide consists of the transmembrane region and part of the cytoplasmic domain of the H2-Kk heavy chain. At steady state, 25% of the total cell surface H2-Kk possesses this modification. In addition, the modification is mutually exclusive with the phosphorylation of the H2-Kk heavy chain at Ser-333. The possible biological significance of the novel modification of class I antigens is discussed.

Amino Acid Sequence↗

PubMed2000

Exposure and acute exposure-effects before and after modification of a contaminated humidification system in a synthetic-fibre plant.

OBJECTIVE: Follow-up study of exposure and acute exposure-effects after modification to steam humidification of a contaminated cold water system which had caused an outbreak of humidifier fever in a synthetic-fibre plant. METHODS: Before and after modification of the system aerobiological measurements were performed. Concentrations of fungi and bacteria, in colony forming units (cfu) per m3, were measured by stationary air sampling with an Andersen sampler. Endotoxin levels (pg/m3) were determined by Limulus Amoebocyte Lysate (LAL) assay in pooled dust from personal air sampling. An indication of exposure levels of oil-mist was obtained by monitoring with a direct reading optical photometer. Changes as acute exposure-effects in spirometry and white blood cell count, during an afternoon shift were compared in exposed and non-exposed workers before and after modification. RESULTS: Measured levels of fungi, total bacteria, Gram-negative bacteria and endotoxins both before and after modification were below levels which would be expected to be associated with the exposure-effects. However, after modification, we found that the statistically significant differences in levels of bacteria and endotoxins with a department without humidification no longer existed. Mean oil-mist concentrations were below 1 mg/m3, with short-time peak exposure during certain tasks of up to 5 mg/m3. Before modification, in exposed workers there was significantly more decline of spirometry, and more increase of white blood cell count during the first afternoon shift, compared with non-exposed workers. In exposed workers, the white blood cell count increase was positively associated with decline of spirometry. After modification, differences between exposed and non-exposed workers no longer existed. CONCLUSION: Follow-up investigation of acute exposure-effects demonstrated the effectiveness of remedial actions taken against a contaminated humidification system. Follow-up of exposure-effects in particular is recommended when there is doubt about the interpretation of exposure measurements.

Bacteria↗