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PubMed1979

High mobility group nonhistone chromosomal proteins also exist in Tetrahymena.

High mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25 M HCl from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5 M HClO4, 0.35 M NaCl, or 4 mM spermidine, together with H1 histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.

Amino Acids↗
PubMed1982

Studies on the serine proteases associated with rat liver chromatin.

The chromatin fraction of rat liver exhibited proteolytic activity caused by serine proteases, with maximal activity at pH 8 or 10. They were analyzed by affinity labeling with [3H]diisopropylfluorophosphate followed by SDS-polyacrylamide gel electrophoresis, and partially purified by gel filtration through Sepharose 6B after selective extraction from the chromatin fraction. The following results were obtained. 1. The chromatin fraction contained three DFP-binding proteins with molecular weights of 52,000, 25,000, and 15,000 daltons, and they were tentatively designated proteins A, B, and C, respectively. Unlike proteins A and B, protein C reacted with DFP more strongly at pH 10 than at pH 8. A greater part of protein B was present in the nucleoli, while the others were predominantly distributed in extra-nucleolar chromatin. 2. Proteins A and B were extracted from the chromatin fraction by 5 M urea and 0.7 M NaCl, respectively; while protein C was not extractable by either solution. Proteins A and B were further purified by gel filtration through Sepharose 6B. 3. Protein B was a neutral protease with a maximal activity for 3H-labeled ribosomal proteins at pH 8 and showed high specificity for basic proteins, such as histone and ribosomal proteins. Protein A was an alkaline protease with a maximal activity at pH 10 and showed proteolytic activity not only for basic proteins but also for hydrophobic proteins, such as casein and non-histone proteins of chromatin. 4. Protein A was activated at pH 8 by high concentrations of NaCl, suggesting the presence of some inhibitor(s). Protein A was converted to protein C at pH 10, and also at pH 8 with high concentrations of NaCl. Thus, protein A is suggested to be the complex of protein C and unknown inhibitor(s). 5. When the chromatin fraction was incubated at pH 10, non-histone proteins were degraded much faster than at pH 8, although H1 histone was degraded at similar rates at both pHs. These results indicate that the chromatin fraction of rat liver contains at least two kinds of serine proteases, B and C, and that protease B is probably involved in the metabolism of basic protein, especially H1 histone. Protease C, the greater part of which associates with some inhibitors to form protein A, may play its main role in the metabolism of non-histone proteins.

Animals↗
PubMed2001

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis.

Acyltransferases↗
PubMed1996

The 32 kDa major outer-membrane protein of Pasteurella multocida capsular serotype D.

The major outer-membrane protein (MOMP) of Pasteurella multocida serotype D strain P210, with an apparent molecular mass of 32 kDa, was purified and characterized. The purification method involved selective extraction of MOMP with N-lauroylsarcosine and SDS, followed by immunoaffinity chromatography using a murine monoclonal antibody (mAb). The N-terminal sequence and amino acid composition of the MOMP showed considerable similarity to other Gram-negative bacterial porins, notably to the 37 kDa MOMP (porin H) of P. multocida. Immunoelectron microscopy and colony blotting assays were used to demonstrate the surface localization of the 32 kDa MOMP on bacterial cells. The colony blotting assay provided a simple, sensitive and rapid screening method for visualizing accessibility of the antibody on the cells. In a Western blot assay, murine polyclonal hyperimmune serum against the purified 32 kDa MOMP recognized both serotype B and D strains bearing either a 32 kDa or a 37 kDa MOMP, whereas the mAb recognized only serotype D strains bearing a 32 kDa but not a 37 kDa MOMP. The present data indicate that the 32 kDa MOMPs of P. multocida are antigenically heterogeneous and possess both specific and cross-reacting epitopes. Detection of type-specific epitopes on the 32 kDa MOMP using an mAb may have potential implications regarding the feasibility of developing a serotyping system for P. multocida.

Amino Acid Sequence↗
PubMed1970

On the relationship between ribulose diphosphate carboxylase and protochlorophyllide holochrome of Phaseolus vulgaris leaves.

The relationship between ribulose diphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase [dimerizing], EC 4.1.1.39, formerly known as carboxydismutase) and protochlorophyllide holochrome of etiolated Phaseolus vulgaris leaves has been studied.A procedure for partially selective extraction of the two proteins was devised using tris-HCl buffer first without and then with Triton X-100. Ribulose diphosphate carboxylase was readily extracted from etiolated bean leaves without Triton X-100, and protochlorophyllide holochrome was extracted on the addition of Triton X-100. Optimal extraction conditions for protochlorophyllide holochrome have been found to be different for tissues of different ages.

Carboxy-Lyases↗
PubMed1976

Extractant Influence on the Relationship between Extractable Proteins and Cold Tolerance of Alfalfa.

The influence of ionic composition and pH of extractant on the relationship between the extracted proteins and the cold tolerance of Vernal and Arizona Common alfalfa (Medicago sativa L.) was examined. Five environments were used to induce different tolerance levels. The quantity of protein extracted from plants was influenced by the hardening environment, cultivar, and ionic composition and pH of 29 extractants. Extractants with a pH below 6 generally extracted less protein.The measured cold tolerance of the plants was correlated with the quantity of protein detected in many of the 14 regions of the electrophoresis gel columns regardless of extractant but was most closely associated with the protein in either region 7 or 8 with nine of ten extractants.The magnitudes of cultivar and hardening effects on quantities of protein detected on various column regions were influenced by choice of extractant. This suggests that proper extractant selection may be vital for study of isozymes by electrophoresis.

Journal Article↗
PubMed1982

Phosphorylation of the adenosine triphosphatase in a deoxycholate-treated plasma membrane fraction from corn roots.

The ATP phosphohydrolase (ATPase) activity of a corn (Zea mays L., WF9 x Mo17) root plasma membrane fraction was enriched almost 2-fold by selective extraction with 0.1% (w/v) deoxycholate. The detergent treatment solubilized about 30% of the total membrane protein and some ATP hydrolyzing activity that was not K(+)-stimulated, but the major portion of the ATPase activity could be pelleted with membranes. The properties of the ATPase associated with the detergent-extracted plasma membrane fraction were similar to those for the ATPase of the untreated plasma membrane fraction with respect to substrate specificity, pH optimum, kinetics with MgATP, ion stimulation, and inhibitor sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only minor differences in protein composition resulting from the detergent treatment.The plasma membrane fraction from corn roots contained an endogenous protein kinase activity. This was shown by the time course of phosphate incorporation and by the labeling of a number of protein bands on SDS-polyacrylamide gel electrophoresis. The deoxycholate treatment removed measurable protein kinase activity and allowed the demonstration of a rapidly turning over covalent phosphorylated intermediate associated with the detergent-extracted plasma membrane fraction. The phosphorylated intermediate was present as a 100,000 dalton polypeptide and may represent the catalytic subunit of the plasma membrane K(+)-ATPase.

Journal Article↗
PubMed1989

Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata).

The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (-)-carvone which is formed by cyclization of the C(10) isoprenoid intermediate geranyl pyrophosphate to the olefin (-)-limonene, hydroxylation to (-)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates.

Journal Article↗
PubMed2005

From projection pursuit and CART to adaptive discriminant analysis?

While many efforts have been put into the development of nonlinear approximation theory and its applications to signal and image compression, encoding and denoising, there seems to be very few theoretical developments of adaptive discriminant representations in the area of feature extraction, selection and signal classification. In this paper, we try to advocate the idea that such developments and efforts are worthwhile, based on the theorerical study of a data-driven discriminant analysis method on a simple--yet instructive--example. We consider the problem of classifying a signal drawn from a mixture of two classes, using its projections onto low-dimensional subspaces. Unlike the linear discriminant analysis (LDA) strategy, which selects subspaces that do not depend on the observed signal, we consider an adaptive sequential selection of projections, in the spirit of nonlinear approximation and classification and regression trees (CART): at each step, the subspace is enlarged in a direction that maximizes the mutual information with the unknown class. We derive explicit characterizations of this adaptive discriminant analysis (ADA) strategy in two situations. When the two classes are Gaussian with the same covariance matrix but different means, the adaptive subspaces are actually nonadaptive and can be computed with an algorithm similar to orthonormal matching pursuit. When the classes are centered Gaussians with different covariances, the adaptive subspaces are spanned by eigen-vectors of an operator given by the covariance matrices (just as could be predicted by regular LDA), however we prove that the order of observation of the components along these eigen-vectors actually depends on the observed signal. Numerical experiments on synthetic data illustrate how data-dependent features can be used to outperform LDA on a classification task, and we discuss how our results could be applied in practice.

Algorithms↗
PubMed1992

Effects of topical retinoids on cytoskeletal proteins: implications for retinoid effects on epidermal differentiation.

In vivo effects of retinoids on epidermal differentiation were investigated by analyzing cytoskeletal proteins in rhino mice treated topically with all-trans-retinoic acid (RA) and other retinoids (13-cis-retinoic acid, etretinate, TTNPB). Non-disulfide-linked cytoskeletal proteins, including keratins from the epidermal "living layers," were first selectively extracted using 9.5 M urea; subsequently, keratins of the stratum corneum were isolated using 9.5 M urea plus a reducing agent. Gel electrophoresis and immunoblot analysis showed that urea extracts of epidermis from vehicle-treated skin were composed predominantly of four major keratins (analogous to human epidermal keratins K1, K5, K10, and K14), and the keratin filament-associated protein filaggrin. In contrast, extracts of epidermis from retinoid-treated skin contained additional keratins (K6, K16, and K17) and almost no detectable filaggrin. Furthermore, similar analysis of stratum corneum keratins demonstrated that extracts from RA-treated skin did not contain the partially proteolyzed keratins typically observed in stratum corneum extracts of control animals. Hyperplasia-inducing agents (salicylic acid, croton oil) caused an increase in keratins K6, K16, and K17, but they did not effect filaggrin or alter proteolysis of stratum corneum keratins. The result that RA induced expression of keratins K6, K16, and K17, as commonly expressed in hyperproliferative epidermis, is consistent with the notion that retinoids increase epidermal cell proliferation in the basal and/or lower spinous layers. The findings that topical RA decreased filaggrin expression and reduced proteolysis of stratum corneum keratins, despite increased size and number of granular cells and the presence of an anucleate stratum corneum, suggest that topical RA may also modulate a later stage of epidermal differentiation involved in stratum corneum formation.

Administration, Topical↗
PubMed1990

Detection of proteins that recognize platinum-modified DNA using gel mobility shift assay.

Using a gel mobility shift assay, we have identified proteins, in the nuclear extracts of a human lung cancer cell line, that recognize cis-diamminedichloroplatinum(II) (cis-DDP, CDDP)-modified DNA. A 158-base-pair double-stranded DNA fragment, derived from pBR322 plasmid DNA, was modified by either CDDP, tetrachloro(dl-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) or trans-DDP (the stereoisomer of CDDP and clinically ineffective). These platinum drug-modified probes were incubated with nuclear extracts and analyzed by gel mobility shift assay. Proteins in the extracts selectively recognized the clinically active platinum-modified DNA fragment. No binding to the trans-DDP-modified DNA fragment was observed. These proteins may play a role in the cytotoxicity or in a DNA repair process.

Cisplatin↗
PubMed2000

p53 mutations in prostatic intraepithelial neoplasia and concurrent carcinoma: analysis of laser capture microdissected specimens from non-transition and transition zones.

Prostatic intraepithelial neoplasia (PIN) is characterized by intraluminal proliferation of epithelial cells and is divided into high-grade (HGPIN) and low-grade (LGPIN) lesions. HGPIN is regarded as the most likely precursor of prostatic cancer (PCA). Microdissected DNA selectively extracted from paraffin-embedded sections of 27 cases with PCA were analyzed for p53 mutation in exons 5 - 8 by single-strand conformation polymorphism of polymerase chain reaction-amplified DNA fragments (PCR-SSCP) followed by direct sequencing. These patients received total prostatectomy (27 cases). After a review of histologic sections, DNA was extracted from 193 locations; 111 lesions from 27 cases with HGPIN (75 lesions from non-transition zone and 36 from transition zone), 55 lesions from 27 cases with PCA (30 lesions from non-transition zone and 25 from transition zone), and 27 from 27 benign glands. Analysis revealed 27 mutations of the p53 gene in 24 lesions from 12 cases. Benign glands adjoining PIN and / or PCA had no mutations. All mutations were point mutations: 17 missense, 7 silent, and 2 nonsense. Mutations were detected in 6 cases (22.2%) or 13 of 111 lesions (11.7%) with HGPIN and 8 cases (29.6%) or 11 of 55 lesions (20.0%) with PCA. In a given case, HGPIN and PCA lesions had different p53 mutations from each other, suggesting multiclonal development of prostatic precancerous lesions. The frequency of p53 mutation of PCA was significantly higher in the non-transition zone (33.3%) than in the transition zone (4%), and higher in the stage T3 cases (30.3%) than in the stage T2 cases (4.5%, 1 of 22 lesions) (both P < 0.05). Frequency of p53 mutation of PIN in the non-transition zone (14.7%) was higher than that in the transition zone (5.6%), although the difference was not significant. The frequency rate of p53 mutation in HGPIN close to PCA ( </= 2 mm) was significantly higher (24%) than that in HGPIN lesions > 2 mm from PCA (3%). All these findings indicate that the p53 gene mutations are involved in prostatic carcinogenesis and explain why the non-transition zone is the predominant site of PCA.

Genes, p53↗
PubMed2005

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis.

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.

Animals↗
PubMed1992

The chemical degradation of phenylmercuric nitrate by disodium edetate during heat sterilization at pH values commonly encountered in ophthalmic products.

The stability following heat sterilization (121 degrees C for 15 min) of phenylmercuric (PM) nitrate in the presence of disodium edetate at pH values 5-8 has been investigated by both high-performance liquid chromatography (HPLC) and atomic absorption spectroscopy (AAS). A stability-indicating HPLC method involving formation of the diethylamine-dithiocarbamate complexes of phenylmercuric and mercuric ions was found to suffer a pH-dependent interference from disodium edetate. A second method was therefore also employed involving selective extraction into diethylether of the phenylmercuric ion followed by HPLC of the piperidinedithiocarbamate complex with concomitant analysis of the unextracted mercuric ion by AAS using the cold-vapour technique. An HPLC method was also developed for benzene in the degraded mixtures. The application of these methods to autoclaved solutions containing PM nitrate and disodium edetate demonstrates that under the conditions of heat sterilization the phenylmercuric ion is degraded to mercuric ion and benzene to the extent of 15% at pH 8, 80% at pH 7 and completely degraded at pH 5 and 6.

Chromatography, High Pressure Liquid↗
PubMed1976

Hormone-induced alterations in nonhistone protein. Methylation and phosphorylation in Sciara coprophila.

Methylation and phosphorylation of chromosomal nonhistone protein (NHP) has been demonstrated in the salivary gland cells of diptera [5, 7] and implicated in the control of gene expression [35, 36]. Furthermore, hormones can stimulate methyl and phosphoryl side chain metabolism and thus enhance template activity. Salivary glands from late fourth instar female larvae of Sciara coprophila (cortisone-supplemented and normal diet) were incubated in 3H-uridine (10 muCi/ml), 3H-thymidine (10 muCi/ml), 3H-methyl-methionine (20 muCi/ml), 35S-methionine (10 muCi/ml) and 32P-orthophosphate (1 mc/ml), for varying time periods, to measure RNA synthesis, DNA synthesis, methylation, protein synthesis and phosphorylation, respectively. Following selective extraction of lipid, histone and nucleic acids, glands were prepared for light microscope autoradiography. A more specific labelling pattern, as well as increased grain number on particular bands, interbands and bulbs, was noted on chromosomes from cortisone-fed larvae incubated in 3H-methyl-methionine for 1 min when compared with larvae on the standard diet. Cortisone also increased RNA synthesis and nucleoprotein phosphorylation, but not DNA or protein synthesis. In summary, cortisone enhances the specificity and degree of NHP methylation and phosphorylation at discrete chromosomal loci, i.e. alterations in side chain metabolism which may be responsible for increased RNA synthesis.

Animals↗
PubMed1982

2'5'-Oligoadenylate polymerase activity in the rat small intestine.

2'5'-Oligoadenylates are potent protein synthesis inhibitors: they are synthesized by a polymerase which was first described in interferon-treated cells. This system may also be involved in a normal process of cell proliferation and differentiation, in the absence of any viral infection. The small intestine enterocyte has been investigated as a model to test this hypothesis. The presence of 2'5'-oligoadenylate polymerase activity is demonstrated in the intestinal mucosa of normal adult rats. The distribution of this enzyme, and of the enzymes degrading 2'5' oligoadenylates have been investigated on enterocyte pools selectively extracted, under mild conditions, from the different parts of the rat small intestine. Similar results were obtained with enterocytes extracted from the mucosa of germ-free animals.

2',5'-Oligoadenylate Synthetase↗
PubMed1978

The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.

Adenosine Triphosphatases↗
PubMed1981

Large-scale purification and phosphorylation of a detergent-treated adenosine triphosphatase complex from plasma membrane of Saccharomyces cerevisiae.

A new procedure for large-scale preparation of plasma-membrane-bound ATPase from Saccharomyces cerevisiae is described. The crude membrane fraction is purified by selective extraction with three successive detergents: deoxycholate (0.25 mg/mg protein), Triton X-100 (0.25%) and lysophosphatidylcholine (1 mg/mg protein). These treatments extract the mitochondria and strip the plasma membrane. From 1 kg commercial baker's yeast, 200 mg of plasma membrane proteins are isolated in 2--3 days. Plasma-membrane-bound ATPase of specific activity of 10--13 mumol Pi x min-1 x mg protein-1 is obtained with a yield estimated to 60%. Dodecylsulfate/polyacrylamide gel electrophoresis shows three predominant polypeptides of Mr = 95000, 70000 and 56000 in the purified membrane fraction. The major polypeptide of Mr = 95000 identified as the ATPase subunit is phosphorylated by millimolar concentrations of ATP. The phosphorylated intermediate reaches the steady-state level in less than 100 ms and turns over very rapidly. It is hydrolyzed by hydroxylamine. Its formation is prevented by the ATPase inhibitors vanadate and Dio-9, a plasma-membrane ATPase inhibitor of unknown structure. At least four other membrane proteins are phosphorylated with much slower kinetics, presumably through the action of protein-kinase(s).

Adenosine Triphosphatases↗