Biomedical subjects

M L Chiu

Publications and source records attributed to M L Chiu.

At least 37 records · Page 2Linked to original sources

PubMed1992

Comparison of neutral and cationic myocardial perfusion agents: characteristics of accumulation in cultured cells.

Uptake and washout kinetics of two new neutral lipophilic technetium-99m-labeled boronic acid adducts of technetium tris(dioxime) (BATO complexes) were studied in monolayers of contractile chick heart cells and compared to the cationic myocardial perfusion agents, 99mTc(CNCH2C(CH3)2OCH3)6+ (Tc-MIBI) and 201Tl+. 99mTcCl(CDOH)2(CDO)(BCH3), where CDO = cyclohexanedione dioxime (CDO-MeB), had a 7-fold greater net accumulation than Tc-MIBI and the most rapid unidirectional washout with a fast initial phase and a slower secondary component. Incubation with cationic membrane transport inhibitors or metabolic inhibitors had little or modest influence, respectively, on uptake of these BATO complexes. Studies with NIH 3T3 fibroblasts indicated that the neutral complexes did not show myocyte specific accumulation.

3T3 Cells↗

PubMed1992

Restricted tissue distribution of a 37-kD possible adherens junction protein.

A major polypeptide of M(r) 37,000 was purified from a desmosome-enriched citric acid-insoluble pellet of pig tongue epithelium. The polypeptide was solubilized from the 4-M urea-insoluble pellet with 9 M urea, and extracts were separated by carboxymethyl cellulose and gel filtration chromatography. The 37-kD protein was obtained in milligram quantities as a single band on two-dimensional gels in 30% yield after 21-fold purification from the citric acid-insoluble fraction. The protein is not glycosylated and has a pI of approximately 8.7. Although isolated from a fraction rich in desmosomes, the 37-kD protein is not a desmosomal protein. Indirect immunofluorescence analysis of frozen sections of tongue and other tissues demonstrated that antibodies raised to the 37-kD protein bound only to suprabasal cell layers at punctate regions of the periphery of the cell and was absent from most regions of epidermis, whereas antibodies to desmoplakins I and II, desmosomal proteins, bound similarly but in all epidermal layers. Immunoelectron microscopy localized the 37-kD protein to the cell periphery in regions between, but never in, desmosomes. By immunofluorescence, the 37-kD protein colocalized with actin as well as with vinculin and uvomorulin in oral tissues. Like the 37-kD protein, vinculin and uvomorulin were absent from the basal layer. Based on its appearance, localization, and solubility properties, the 37-kD protein is probably a component of adherens junctions; its restriction to suprabasal cells and exclusion from the epidermis are unique.

Actins↗

PubMed1992

Comparative effects of neutral dipolar compounds and lipophilic anions on technetium 99m-hexakis (2-methoxyisobutyl isonitrile) accumulation in cultured chick ventricular myocytes.

RATIONALE AND OBJECTIVES: Non-flow-dependent myocellular accumulation and uptake kinetics of the myocardial perfusion and viability imaging agent, hexakis (2-methoxyisobutyl isonitrile) technetium 99m(I) (Tc-SESTAMIBI), are thermodynamically driven by large negative sarcolemmal and mitochondrial membrane potentials, and can be enhanced by addition of the lipophilic anion, tetraphenylborate (TPB). To further understand the general properties required of a co-administered compound for increasing the kinetic response of Tc-SESTAMIBI to membrane potential, a systematic appraisal of additional candidate lipid-soluble anions and neutral dipolar compounds was undertaken. METHODS: Each compound was biologically tested for its ability to enhance Tc-SESTAMIBI accumulation in a cultured heart cell model, and electronic dipole moments were evaluated using semi-empirical molecular orbital calculations. RESULTS: Of this series, phloretin (100 microM), TPB (10 microM), and to a lesser degree, 8-anilino-1-naphthalene sulfonate (100 microM) enhanced myocellular accumulation of Tc-SESTAMIBI. Phloretin enhancement was pH-dependent, showing maximal effect at pH 7.4, and was not additive to the augmentation induced by TPB. A series of additional lipid soluble anions and structural analogues of phloretin were without effect. CONCLUSION: Although selected compounds enhanced Tc-SESTAMIBI accumulation, overall, no direct relationship of dipole moment to biologic enhancement was demonstrated.

Anilino Naphthalenesulfonates↗

PubMed1992

Severe gastric mucosal changes following vagotomy with duodenogastric reflux.

We conducted an experimental study of the morphological changes in gastric mucosa following vagotomy, in particular of the chronic effects of duodenogastric reflux. Male Wistar rats were divided into four groups: sham operation, duodenogastric reflux (DGR), truncal vagotomy with duodenogastric reflux (TV + DGR), and truncal vagotomy with bypass (TV + bypass). The last operation was performed to observe the effects of vagotomy without reflux. The duodenogastric reflux procedure was established by cutting the duodenum just distal to the orifice of bile and pancreatic ducts following gastrojejunostomy. After 12 and 30 weeks, animals were killed for morphological studies of gastric mucosa. The TV + DGR group showed formation of multiple elevated lesions macroscopically, and microscopically showed marked atrophy of gastric glands and proliferation of cystically dilated adenomatous lesions, which were regarded as precancerous or paracancerous lesions. The DGR group showed chronic gastric ulcer and hyperplasia of the gastric glands. However, the TV + bypass group showed no remarkable changes in the gastric mucosa. These results indicate that chronic effects of duodenogastric reflux following vagotomy induce severe inflammatory changes as well as structural alteration of the gastric mucosa. Therefore, a regular follow-up observation is necessary after vagotomy when combined with a drainage procedure because the latter could promote duodenogastric reflux and produces pathological changes to the stomach.

Animals↗

PubMed1992

Divergent kinetics of 201Tl and 99mTc-SESTAMIBI in cultured chick ventricular myocytes during ATP depletion.

BACKGROUND: Thallous chloride (201Tl) and hexakis(2-methoxyisobutyl isonitrile) technetium (I) (99mTc-SESTAMIBI) are myocardial perfusion imaging agents with biological properties that also reflect tissue viability. Initial myocellular uptake rates of 201Tl reflect activity of Na,K-ATPase, whereas those of 99mTc-SESTAMIBI reflect mean plasma membrane potential. METHODS AND RESULTS: To better understand the mechanistic responses of these tracers to myocellular injury, cultured chick embryo cardiac myocytes were metabolically inhibited in iodoacetate (1 mM) and rotenone (10 microM) for up to 2 hours, and initial uptake rates of each agent were determined at successive intervals along with correlative cellular contents of ATP, sodium, and potassium and lactate dehydrogenase release. ATP content fell from 30.5 +/- 1.4 to 2.7 +/- 0.9 nmol.(mg protein)-1 within 2 minutes, whereas sodium and potassium contents ran down their thermodynamic gradients more slowly (t 1/2 approximately 60 minutes). Modestly severe cell injury was produced at 2 hours as estimated by lactate dehydrogenase release (18% of total). Initial uptake rates of 201Tl declined from 6.9 +/- 0.8 to 4.0 +/- 0.4 fmol.(mg protein)-1.(nMo)-1.(min)-1 by 20 minutes and remained depressed and ouabain (100 microM)-insensitive at 30 +/- 13% of control. Conversely, initial uptake rates of 99mTc-SESTAMIBI increased from 10.6 +/- 0.8 to 15.0 +/- 0.6 fmol.(mg protein)-1.(nMo)-1.(min)-1 within 10 minutes, remained elevated for 40-60 minutes, and later declined to low values. Injury-induced enhancement of initial uptake rates of 99mTc-SESTAMIBI were insensitive to ouabain (100 microM), carbonyl cyanide-m-chlorophenyl hydrazone (5 microM), and valinomycin (1 microgram/ml) but were significantly inhibited by 130 mM Ko buffer, Ba2+ (1 mM), glybenclamide (100 microM), and quinacrine (10 microM). CONCLUSIONS: Uptake rates of 201Tl monotonically decline, correlating with Na-K pump inhibition from ATP depletion. Conversely, uptake rates of 99mTc-SESTAMIBI at first increase above control for 40-60 minutes, indicating a mean plasma membrane hyperpolarization possibly resulting from opening of ATP-sensitive and arachidonic acid-activated potassium channels, before declining to low values with more severe cell injury. Correlative non-flow-dependent relations between 201Tl and 99mTc-SESTAMIBI contain information regarding the degree of myocellular injury.

Adenosine Triphosphate↗

PubMed1992

Subcellular distribution and analysis of technetium-99m-MIBI in isolated perfused rat hearts.

To address the apparent discrepancy between cultured cells and whole heart preparations, Langendorff-perfused rat hearts loaded with hexakis (2-methoxyisobutyl isonitrile) technetium (I) (99mTc-MIBI) were fractionated by a standard differential centrifugation method and fractional contents of 99mTc-MIBI were correlated with the mitochondrial marker, malate dehydrogenase (MDH), and mitochondrial substrates. The "cytosolic" fraction nominally contained 89% +/- 3% of total 99mTc-MIBI, but also contained 91% +/- 1% of total MDH activity by this method. Chromatographic analysis of activity in the "cytosolic" fraction demonstrated greater than 95% of the agent was present as the original free cationic complex; binding to a small molecular weight cytosolic protein was not involved in localization. Addition of the mitochondrial uncoupler CCCP (5 microM) to both "mitochondrial" and "cell fragment" pellets released up to 84% +/- 8% of 99mTc-MIBI content and addition of the mitochondrial substrate succinate (10 microM) in the presence of rotenone (1 microM) enhanced 99mTc-MIBI content by up to 139% +/- 52% over the control. These correlative data from rat hearts indicate that approximately 90% of 99mTc-MIBI activity in vivo is associated with mitochondria in an energy-dependent manner as a free cationic complex, but migrates during fractionation/centrifugation.

Animals↗

PubMed1991

Distal and proximal ligand interactions in heme proteins: correlations between C-O and Fe-C vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in C17O- and 13CO-labeled species.

We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7----Val E7; His E7----Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C-O vibrational frequencies, v(C-O), and delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) and v(Fe-C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of pi-back-bonding from Fe d to CO pi* orbitals, as outlined previously [Li, X.-Y., & Spiro, T.G. (1988) J. Am. Chem. Soc. 110, 6024]. The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.

Carbon Isotopes↗

PubMed1991

1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation.

The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength. At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1. 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks. The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder. The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures. At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield. Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination. The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond. The contact shifts for a coordinated Met in the high-spin state are estimated. A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations.

Cytochrome b Group↗

PubMed1991

Cervical enamel projections in Chinese first permanent molars.

Cervical enamel projections in relation to molar furcations may act as predisposing factors in periodontal attachment loss. Limited data are available documenting these features in Chinese teeth. The purpose of this study was to determine the prevalence and distribution in a sample of first permanent molars from Hong Kong Chinese. Three hundred and sixty-two extracted first molars (194 maxillary, 168 mandibular) were selected from the Prince Philip Dental Hospital tooth collection and examined using a dissection microscope for identification and classification. Cervical enamel projections were identified in 59 per cent of maxillary and 79 per cent of mandibular first molars. Buccal aspects were more frequently involved. Forty-nine per cent were Grade III (extending to the furcation area); over one-half were particularly slender while 18 per cent were discontinuous. A modification to the classification system is proposed in order to delineate these features in future studies.

China↗

PubMed1991

Enhancement by tetraphenylborate of technetium-99m-MIBI uptake kinetics and accumulation in cultured chick myocardial cells.

Myocellular uptake and retention of technetium-99m-hexakis (2-methoxyisobutylisonitrile) (Tc-MIBI), a lipophilic cationic myocardial perfusion and viability imaging agent, is dependent on both mitochondrial and plasma-membrane potentials. To test for enhancement of uptake kinetics by lipophilic anions, cultured chick heart cells were exposed to tetraphenylborate (TPB), which produced a concentration-dependent maximal 15-fold increase in Tc-MIBI uptake kinetics (at 3 x 10(-5) M) and enhanced peak accumulation of Tc-MIBI from 165.4 +/- 26.3 to 705.6 +/- 61.3 fmoles/mg protein.nMo (P less than 0.001). Carbonyl cyanide-m-chloro phenylhydrazone (CCCP; 10(-5) M), a mitochondrial uncoupler, rapidly depleted cellular content of Tc-MIBI in the presence of TPB (10(-5) M) from 300.0 +/- 30.0 to 42.5 +/- 1.9 fmole/mg protein.nMo (p less than 0.001). TPB enhanced both uptake rates and net accumulation of Tc-MIBI at all buffer Ko concentrations between 130 mM and 0.54 mM. Tc-MIBI influx rates allowed estimation of plasma-membrane potential as a function of Ko in the presence of valinomycin with a slope of -67 mV/decade (r = -0.99). The results further support a potential-dependent mechanism for cell uptake of Tc-MIBI and suggest a rational approach for increasing tissue extraction fraction in vivo.

Animals↗

PubMed1990

Uptake and retention of hexakis (2-methoxyisobutyl isonitrile) technetium(I) in cultured chick myocardial cells. Mitochondrial and plasma membrane potential dependence.

The fundamental myocellular uptake and retention mechanisms of hexakis (2-methoxyisobutyl isonitrile) technetium(I) (Tc-MIBI), a technetium-99m-based myocardial perfusion imaging agent, are unresolved. Because of the lipophilic cationic nature of Tc-MIBI, it may be distributed across biological membranes in response to transmembrane potential. To test this hypothesis, net uptake and retention of Tc-MIBI in cultured chick embryo ventricular myocytes were determined under conditions known to alter mitochondrial and plasma membrane potentials. Isovolumic depolarization of plasma membrane potentials in 130 mM extracellular K (Ko) 20 mM extracellular Cl buffer reduced net accumulation of Tc-MIBI from 171 +/- 16 (control) to 29 +/- 3.3 fmol intracellular Tc-MIBI/mg protein.nM extracellular Tc-MIBI. Unidirectional influx of Tc-MIBI in cells depolarized in 30 mM Ko buffer was also reduced; a resting plasma membrane potential of -87 +/- 6 mV was calculated from the Goldman flux equation using normal Ko/high Ko Tc-MIBI influx ratios. Addition of the potassium ionophore valinomycin to cells incubated in 130 mM Ko buffer to additionally depolarize mitochondrial membrane potentials further reduced net uptake of Tc-MIBI to levels comparable to that found in nonviable freeze-thawed preparations ([Tc-MIBI]i/[Tc-MIBI]o = 1). By depolarizing mitochondrial (and in part plasma membrane) potentials with the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone (CCCP) Tc-MIBI was rapidly depleted from 181 +/- 16 (control) to 16 +/- 2.6 and 31 +/- 4.2 fmol/mg protein.nMo, respectively, with kinetics that did not correlate with loss of cellular ATP content. CCCP alone inhibited 90 +/- 3% of net accumulation or 66 +/- 3% of unidirectional influx of Tc-MIBI in a concentration-dependent manner. By hyperpolarizing mitochondrial membrane potentials with the K+/H+ ionophore nigericin or the ATP synthase inhibitor oligomycin, net uptake and retention of Tc-MIBI were increased by 60 +/- 9% and 375 +/- 20%, respectively. Caffeine, as well as the respiratory chain electron transport inhibitor rotenone, did not significantly alter net cell uptake (p greater than 0.2). These data indicate that the fundamental myocellular uptake mechanism of Tc-MIBI involves passive distribution across plasma and mitochondrial membranes and that at equilibrium Tc-MIBI is sequestered within mitochondria by the large negative transmembrane potentials.

Animals↗

PubMed1990

Effect of mitochondrial and plasma membrane potentials on accumulation of hexakis (2-methoxyisobutylisonitrile) technetium(I) in cultured mouse fibroblasts.

Hexakis(2-methoxyisobutylisonitrile) technetium(I) (Tc-MIBI) is representative of a class of 99mTc-based lipophilic cationic myocardial perfusion imaging agents. To test the hypothesis that the mechanism of cellular uptake may involve distribution across biologic membranes in response to membrane potential, Tc-MIBI net uptake and retention were determined in cultured mouse BALB/c 3T3, NIH 3T3, and v-src transformed NIH 3T3 fibroblasts as well as in cultured chick embryo heart cells. Isovolumic depolarization of plasma membrane potentials with 130 mM K 20 mM Cl buffer decreased Tc-MIBI net cell uptake in all preparations. In BALB/c 3T3 cells, depolarizing mitochondrial membrane potential with valinomycin in high K buffer or with the protonophore CCCP inhibited net uptake and retention of Tc-MIBI while hyperpolarizing mitochondrial and plasma membrane potentials with the K+/H+ exchanger nigericin increased Tc-MIBI net uptake. These results indicated that net cellular uptake and retention of Tc-MIBI in fibroblasts were determined by both mitochondrial and plasma membrane potentials; the gamma-emitting properties of Tc-MIBI may therefore raise the possibility of monitoring membrane potential in vivo.

Animals↗

PubMed1989

Placental keratinocyte growth factor: partial purification and comparison with epidermal growth factor.

A water-soluble extract of term human placenta, which was previously shown to promote proliferative growth of human keratinocytes in defined medium, enhanced both cellular attachment and proliferative growth. We have partially purified the activity which enhanced cell growth and examined its action in keratinocytes. Activity was precipitated from the crude extract by (NH4)2SO4 between 33 and 60% saturation and chromatographed by gel filtration. The activity did not bind to heparin-Sepharose at low ionic strength but was adsorbed to DEAE-cellulose from which it was eluted with NaCl and then passed over phenyl-HPLC to remove bovine serum albumin previously added to protect the activity. The active fraction was applied to gel exclusion HPLC in the presence of 0.02% octyl-beta-D-glucopyranoside, which yielded an apparent Mr 35,000 for the factor. Purification was approximately 200-fold with approximately 4% recovery. The factor appears to be a protein, since activity is destroyed by trypsin. Autoradiography of cultures treated with the placental factor or epidermal growth factor (EGF) revealed that approximately 50% of cells were labeled after treatment with either growth factor compared to 9% in control cultures after a [3H]thymidine pulse. Protein synthesis was increased by about 50% 42 h after treatment with either agent, consistent with a 50% increase in nuclear labeling. Cell number was increased fivefold after 6 days in the presence of the partially purified factor, whereas EGF increased cell number eightfold. Stimulation of [3H]thymidine incorporation by the partially purified factor, in contrast, was about twice that produced by EGF, indicating that thymidine incorporation is preferentially stimulated by the placental factor and does not correlate well with other parameters of proliferative growth. The placental keratinocyte growth factor is a unique factor with a novel effect on incorporation of thymidine into DNA.

Cell Division↗

PubMed1988

Stimulation of thymidine incorporation in keratinocytes by insulin, epidermal growth factor, and placental extract: comparison with cell number to assess growth.

The results of a thymidine incorporation assay were compared with direct measurement of cell number in assessment of proliferative growth of human keratinocytes in monolayer culture. Keratinocytes were cultured in supplemented MCDB 153 medium in 0.1 mM Ca2+, and plated in 24-well trays. The ability of insulin, placental extract, and epidermal growth factor to enhance growth and thymidine incorporation were compared. Autoradiography was performed to determine the percentage of cells with labeled nuclei. Epidermal growth factor increased thymidine incorporation under the conditions of the assay, and placental extract increased incorporation by up to 50-fold, since the control cells plated in the absence of epidermal growth factor and other growth factors survived but proliferated minimally. Both cell number and thymidine incorporation showed similar concentration dependence upon insulin and placental extract. If placental extract was added to cells plated 28 h earlier, incorporation was maximal after 17 h in the presence of the extract. If cells were plated in the presence of the extract, 85% of nuclei were shown by autoradiography to be labeled after 23 h, but only 24% of nuclei were labeled in the absence of the extract. A plating density of 10(4) cells/2-cm2 well was optimal. The assay permits rapid identification of growth-promoting fractions without prolonged growth periods, and is a valid indicator of these agents in keratinocyte cultures.

Autoradiography↗

PubMed1988

Stimulation of growth of keratinocytes by basic fibroblast growth factor.

Numerous heparin-binding growth factors active in different types of cells have recently been shown to belong to the family of fibroblast growth factors. Because these factors are active in some types of epithelial cells, we tested the activity of basic fibroblast growth factor (bFGF) from bovine brain in human keratinocyte cultures. bFGF stimulated thymidine incorporation and cellular proliferation in these cultures with half-maximal activity at approximately 60 pg/ml (4 X 10(-12) M). Stimulation of thymidine incorporation was associated with increased nuclear labeling after 22 h in the presence of bFGF under the same conditions used in the thymidine incorporation assay. bFGF was nearly as effective as epidermal growth factor (EGF) in stimulating keratinocyte growth and substantially less effective than crude placental extract, and was not additive with EGF in stimulating thymidine incorporation or proliferation of cells. The findings indicate that bFGF is a potent growth factor for keratinocytes.

Cell Division↗

PubMed1986

Absence of histone H2B in nucleosomes containing histone TH2B and interaction of immunoglobulin with nucleosomes.

Nucleosomes containing histone TH2B were isolated from chromatin subunits of rat testis nuclei (MNT) by incubating with anti-TH2B immunoglobulin (IgTH2B) which was covalently attached to agarose gels. Electrophoretic separation of histones of these isolated nucleosomes revealed that histone H2B was completely absent, suggesting that histone TH2B, the variant of H2B, existed in nucleosomes only as TH2B X TH2B and that TH2B X H2B was not likely to exist in chromatin. Sucrose gradient ultracentrifugation of mixtures of MNT and IgTH2B revealed that when excess amounts of immunologically active IgTH2B were present, complexes of higher sedimentation coefficients than MNT X IgTH2B were formed, but with limited amounts of active IgTH2B, only MNT X IgTH2B was formed. When purified IgTH2B was coated on polystyrene tubes and incubated with MNT, those MNT immobilized by the tube-coated IgTH2B adsorbed IgTH2B from diluted antiserum during subsequent incubation. Those results suggested the absence of steric hindrance in the binding of IgTH2B to MNT X IgTH2B. When MNT was coated on polystyrene tubes and incubated with DNase and then with dilute anti-TH2B antiserum, it was found that DNase digestion increased the binding of immunoglobulin to the tubes approximately 76%. Interaction of chromatin subunits of rat liver nuclei (MNL) with anti-TH2B antiserum was negligible, but DNase digestion of MNL coated on tubes was followed by considerable interaction with anti-TH2B antiserum. Those results indicated DNase unmasked at least part of the determinants encased by DNA. Anti-H2B immunoglobulin (IgH2B) interacted with histone H2B and TH2B to the same extent, and interacted significantly to a lesser extent with either MNT or MNL. DNase digestion of MNT and MNL increased binding of IgH2B approximately 170 and 117%, respectively.

Animals↗

PubMed1985

Effect of inhibition of DNA synthesis on histone synthesis, turnover, and deposition in the rat testis.

In testicular seminiferous epithelial cells (SEC) of normal and hypophysectomized rats, 1-beta-D-arabinofuranosylcytosine and hydroxyurea (at concentrations which inhibited DNA synthesis nearly completely) inhibited histone synthesis only partially, and to a different extent for each histone fraction. In the presence of the inhibitors, the extent of synthesis relative to the corresponding control was TH1-x greater than H1 greater than TH2B-x = X2 = H2A greater than H2B = H3 greater than H4, in which synthesis of the H4 fraction was about 50% of control and that of TH1-x was 90-95% of control. The extent of inhibition of synthesis of each histone fraction was similar after hypophysectomy and, therefore, the changing of the relative populations of heterogeneous cells in the SEC did not influence the relative effects of the inhibitors of DNA synthesis on the synthesis of the various histone fractions. After [3H]leucine injection, the molar proportions of labeled histones relative to H4 decreased markedly between 1.5 h and 6-15 days; this finding indicated that there was rapid removal of histones compared to the H4 fraction during this period. When [14C]thymidine was injected 24 h prior to hydroxyurea treatment and [3H]leucine injection, the ratios of specific activities of histone H4 to DNA did not change significantly over an 11-day period. It appears that newly synthesized histone H4 and other somatic histones are associated with existing DNA in the presence of DNA inhibitors.

Animals↗

PubMed1984

Isolation of rat testis histone TH2B-x, and interaction of TH2B-x antiserum with histones and mononucleosomes.

A method is reported for the isolation of histone TH2B-x from rat testis by affinity chromatography on an agarose-p-chloromercurianilino column. This purified TH2B-x was used to raise antibodies in the rabbit, and the antiserum was assayed by an enzyme-linked double-antibody procedure. At low concentration the antiserum cross-reacts with histone H2B and with histones TH1-x + H1 to the extent of 11-14% of the interaction with TH2B-x. Antiserum preincubated in three successive H2B-coated tubes still retains 80-89% of the original anti-TH2B-x activity when assayed subsequently in TH2B-x-coated tubes, but cross-reaction with H2B is practically zero. The anti-TH2B-x antibodies also interact with tubes coated with mononucleosomes isolated from nuclei of seminiferous epithelial cells (SEC) of rat testis, but the interaction with mononucleosomes from rat liver nuclei is almost zero. The data suggest that in nucleosomes some of the antigenic determinants which are unique to TH2B-x are accessible, while those determinants which are common to H2B and TH2B-x are not accessible for interaction with antibodies. Competition by mononucleosomes, both from rat testis SEC and rat liver (to a lesser degree), in solution is detected by the reduction of binding of enzyme-labeled IgG to TH2B-x-coated tubes. However, an attempted competition by histones TH2B-x or H2B in solution resulted in an increase in the binding of the enzyme-labeled IgG to the mononucleosome-coated tubes. The interpretation of this type of competition assay is complicated by possible interaction of added histones with the coating mononucleosomes, followed by binding of antibodies to the histones. This TH2B-x antibody should be useful in studying changes in structure and function of chromatin during spermatogenesis and in the isolation of TH2B-x mRNA.

Animals↗