Choh Hao Li: April 21, 1913 - November 28, 1987.
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Biochemistry↗
Biomedical subjects
R D Cole
Publications and source records attributed to R D Cole.
Prognostic significance of skin involvement from mucosal tumors of the head and neck.
OBJECTIVE: To examine the hypothesis that skin involvement from mucosal squamous cell carcinoma of the head and neck is a prognostic indicator of a poor outcome. DESIGN: Retrospective review of cases and statistical assessment of median survival times. PATIENTS: Patients with mucosal squamous cell carcinoma of the head and neck. Fifteen patients had direct skin extension and 11 patients had intradermal lymphatic spread. SETTING: University medical center. RESULTS: Direct skin involvement was a prognostic sign of poor outcome but was less ominous than skin involvement by intradermal lymphatic spread. The patients with direct involvement had a 7-month median survival; those with lymphatic spread had a 3-month median survival. At 3 years, all but one patient had died. Involvement of facial skin was better prognostically for duration of survival than was involvement of neck skin. Surgical resection of the involved skin in half of the patients extended palliation 20 months beyond the median survival of the other patients. CONCLUSIONS: Skin involvement from mucosal squamous cell carcinoma of the head and neck indicates a poor prognosis, but resection offers short-term palliation.
Carcinoma, Squamous Cell↗
Effect of pH on interactions between DNA and high-mobility group protein HMG1.
Fluorescence quenching was used to test the effect of pH changes on the binding of high-mobility group protein 1 (HMG1) to double-stranded and single-stranded DNA. At pH 7.5, the binding constant K for double-stranded DNA was 3 x 10(6) M-1, the binding site size n was 13, and the cooperativity factor q was 78, while at pH 6 the corresponding values were K = 12 x 10(6) M-1, n = 54, and q = 770. For the binding of HMG1 to single-stranded DNA at pH 7.5, the values were K = 2 x 10(6) M-1, n = 7, and q = 60, whereas at pH 6 they were K = 3 x 10(6) M-1, n = 14, and q = 440. Denaturation of HMG1 by oxidation of its sulfhydryl groups substantially affected the binding parameters. At pH 6, double-stranded DNA bound oxidized HMG1 with K = 6 x 10(6) M-1, n = 16, and q = 200, and single-stranded DNA bound with K = 3 x 10(6) M-1, n = 7, and q = 180. The sensitivity of the double-stranded DNA-HMG1 interaction to pH, along with an earlier report of a sharp optimum of binding at 140 mM NaCl, reveals a potential for in vivo regulation of the strength and mode of HMG1 binding by DNA through the action of analogous factors in the cellular milieu.
DNA↗
Effects of various salts and pH on the stability of the nucleosome in chromatin fragments.
The stability of nucleosomes in long chromatin fragments was observed by differential scanning calorimetry over a wide range of solution conditions. The thermal denaturation of chromatin was characterized in general as three major transitions, although the process clearly is more complex. The three major transitions were (1) denaturation of the nucleosome, (2) base unstacking of DNA in the resulting denatured nucleoprotein, and (3) base unstacking of naked DNA. In very low salt concentrations (e.g., 2 mM sodium cacodylate), these three processes were essentially coincident (near 76 degrees C), but in medium salt concentrations (e.g., 100 mM NaCl) the nucleosome denaturation occurred first at about 69 degrees C and then base unstacking occurred at 85 degrees C. As [NaCl] was increased, all three processes were resolved with the observation of increasing amounts of naked DNA being melted, until at 2000 mM NaCl the calorimetric profile showed mainly the melting of DNA. The transition temperature for nucleosome denaturation decreased from 76 to 63 degrees C as the salt concentration increased from 1 to 600 mM. Destabilization of the nucleosome by increasing [NaCl] was also evident above 100 mM as a decrease in enthalpic change attributable to nucleosome denaturation. Similarly, as [NaCl] was increased above 100 mM, less and less denatured nucleoprotein was evident as more and more of the DNA melted as naked DNA. The fatty acid salts, sodium valerate and sodium caproate, destabilized the nucleosome but not the denatured nucleoprotein that resulted from the collapse of the nucleosome. In the series acetate, butyrate, valerate, caproate, it was clear that destabilization of the nucleosome increased as hydrophobicity (chain length) increased.(ABSTRACT TRUNCATED AT 250 WORDS)
Calorimetry, Differential Scanning↗
Selectivity in the interaction of various DNA sequences with H1 histone.
An assay for the binding of H1 histone by DNA was developed based on extraction with phenol, which partitions free DNA into the aqueous layer and aggregates of H1 histone-DNA complexes into the phenol layer and interface. When this assay was performed on fragments of simian virus 40 (SV40) DNA, fragments containing the 21-bp repetitive element and a portion of the origin of replication were resistant to H1 binding. This result was corroborated when an endonuclease protection assay showed that the origin was poorly protected by H1 compared to other sites. DNase I protection mapping demonstrated that H1 "underprotected" sites immediately to either side of the AT element, which lies in the origin of replication. These sites were also hypersensitive to attack by hydroxyl radical in the absence of histone, probably indicative of some conformation aberration such as minor-groove distension. The same DNA sequences resistant to binding H1 histone resisted binding to H4 histone but showed much less selectivity, if any, in binding polylysine. These results clearly demonstrate that the interaction of DNA and H1 (and H4 histone) is more complicated than just charge neutralization and probably involves the conformation of the DNA.
Animals↗
Specific interaction between H1 histone and high mobility protein HMG1.
High mobility group proteins HMG1 and -2 and histone H1 are structural components of chromatin. Previously, we reported that HMG1 interacts with H1 histone in a way that modulates the ability of H1 to condense DNA in vitro, suggesting that these proteins may act together in vivo to regulate locally the condensation state of chromatin, possibly affecting replication and/or transcription. Here we show that reduced (native) HMG1 binds to H1 cooperatively at pH 6.0 as a tetramer with a dissociation constant of 3.4 x 10(-8) M, and at pH 7.5 as a monomer with a dissociation constant less than 10(-9) M. Denaturation through oxidation of sulfhydryl groups has a strong effect on the interaction of HMG1 with H1 histone, suggesting that the reduced state of HMG1 is critical to its function. Oxidized HMG1 failed to bind H1 at pH 7.5, and its binding at pH 6 was biphasic; the first three (or two) molecules of H1 were bound with a dissociation constant of 2 x 10(-8) M with negative cooperativity, and the last one (or two) H1's were bound cooperatively with KD = 1.8 x 10(-7) M. Regulation of the pH or the concentration of some other ion may be used in vivo to alter the interactions between HMG1 and -2, H1 histone, and DNA.
Animals↗
Aberrant internal carotid artery.
An aberrant internal carotid artery (ICA) in the middle ear is rare. If unrecognized, it may lead to massive hemorrhage or other catastrophic results during even a minor surgical procedure of the tympanic membrane or middle ear. Recognition of this entity is particularly significant to the primary care or emergency department physician who performs tympanocentesis as a routine part of clinical practice. We present the management and radiographic findings of a case of aberrant ICA referred because of persistent bleeding following a minor surgical procedure. The possibility of an anomalous artery in the middle ear had been recognized during surgery. The procedure was aborted and the patient referred for definitive care.
Carotid Artery, Internal↗
Gunshot wounds to the mandible and midface: evaluation, treatment, and avoidance of complications.
As American society becomes progressively violent, an ever-increasing number of gunshot wounds are being seen across the United States. Particularly challenging are injuries that involve the mandible and midface, not only because of problems with reconstructing bone and soft-tissue defects but also because of emergent problems with airway obstruction and neurovascular compromise. We present 40 cases of gunshot wounds to the mouth, mandible, and maxilla treated at Wake Forest University Medical Center during the past 7 years. The focus of this retrospective analysis is on emergency evaluation and treatment, complications encountered, and operative techniques used for reconstruction. Special emphasis is placed on recognizing and avoiding the complications of these injuries.
Adolescent↗
Histone H1 in modulation of chromatin transcriptional activity.
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Animals↗
Partial displacement of histone H1 from chromatin is required before it can be phosphorylated by mitotic H1 kinase in vitro.
The massive nonselective and reversible phosphorylation of histone H1 during mitosis is a universal phenomenon among eukaryotes. The growth-associated kinase responsible for this phosphorylation is identical to the maturation promoting factor, a key regulator of the cell cycle. Here we showed that growth-associated kinase, isolated from mitotic HeLa cells which were capable of phosphorylating HeLa H1 in vitro with high activity and mostly at the same sites phosphorylated during mitosis in vivo (assayed by two-dimensional analysis of tryptic phosphopeptides), did not significantly phosphorylate chromatin-bound or nuclear H1 in vitro. Its inability to phosphorylate chromatin-bound H1 did not change when the amount of kinase was increased or the incubation was prolonged. The resistance of chromatin-bound H1 to phosphorylation did not result from chromatin aggregation. Rapid phosphorylation of H1 in vitro, as well as in a nuclear system, was restored when NaCl concentrations were raised above 200 mM where H1:DNA interactions are weakened. At 300 mM NaCl, chromatin-bound H1 was phosphorylated in a subset of the sites observed for free H1 phosphorylated in vitro. These results suggest that active displacement of H1 from chromatin DNA may take place before H1 can be fully phosphorylated during mitosis.
Autoradiography↗
Effects of salt concentration and H1 histone removal on the differential scanning calorimetry of nuclei.
The effects of increasing NaCl concentrations on the melting profiles of chromatin in isolated nuclei contradicted published claims that structural transitions near 76 degrees C (Tn-7), near 89 degrees C (Tn-8), and near 105 degrees C (Tn-10) were respectively the melting of linker DNA, the melting of extended nucleosomal strands, and the collapse of nucleosomes in the 300-A fiber. Contrary to expectations of such an interpretation, decreases in salt concentration stabilized Tn-7 and failed to eliminate Tn-10. Moreover, nuclei depleted of H1 histone, which is known to be essential for the formation of the 300-A fiber, gave the same melting profile as intact nuclei with regard to the relative magnitudes of Tn-8 and Tn-10. The effect of salt concentration on the melting profiles and the insensitivity of Tn-8 and Tn-10 to H1 histone removal supports the notion that Tn-7 is the collapse of the nucleosome while Tn-8 and Tn-10 are respectively the unstacking of nucleotide bases in relaxed chromatin and supercoiled chromatin. The identification of Tn-8 as the unstacking of bases in relaxed DNA, and Tn-10 as unstacking in supercoiled DNA, shows that scanning calorimetry can be used to measure the state of repair of DNA in the nucleus. The gain in Tn-8 at the expense of Tn-10 that is seen as the mitotic index drops and differentiation occurs suggests that nicks accumulate in the DNA, perhaps because the gross aggregation of the inactive majority of the chromatin makes it inaccessible to repair enzymes.
Calorimetry, Differential Scanning↗
The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.
To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.
Animals↗
Flanking sequences of Xenopus 5 S RNA genes determine differential inhibition of transcription by H1 histone in vitro. Mitotic phosphorylation of H1 decreases its inhibitory power.
In Xenopus laevis chromatin histone H1 selectively inhibits the transcription of oocyte 5 S RNA genes while not affecting the transcription of somatic 5 S RNA genes (Schlissel, M. S., and Brown, D. D. (1984) Cell 37, 903-913; Wolffe, A. P. (1989) EMBO J. 8, 527-537). To explore possible mechanisms of this specific action of H1 we analyzed the in vitro transcription of H1.DNA complexes. We found that the selective inhibitory effect of H1 in this system depends entirely on the flanking sequences of 5 S RNA genes and not on the coding sequence itself. At an H1:DNA ratio above approximately 0.4, H1 strongly inhibited the transcription of the gene surrounded by the A + T-rich flanks characteristic of oocyte 5 S RNA genes, whereas it did not prevent transcription of the genes surrounded by G + C-rich somatic-type flanks. This was reflected by strongly preferential binding of H1 to isolated 5 S RNA genes contained within A + T-rich flanks. We also showed that superphosphorylation of H1 with growth-associated (mitotic) H1 kinase invariably decreased H1's ability to inhibit transcription in an in vitro system.
Animals↗
A single-column chromatographic system for the analysis and preparation of high mobility group proteins 1 and 2 and other chromosomal proteins using nondenaturing solvents.
One-step chromatography on a Mono S column allows the purification of high mobility group (HMG) proteins 1 and 2 under nondenaturing conditions. Chromatography of HMG1 and -2 on Mono S can be achieved with three of the most widely employed extraction techniques for chromosomal proteins, 0.35 M sodium chloride, 0.74 M perchloric acid, and 0.4 N sulfuric acid. In each case HMG1 and -2 are purified away from the other chromosomal proteins, histone H1, and core histones, and are resolved into both their reduced and oxidized forms. Additionally histone H1 and the core histones are fractionated on Mono S, thus the entire complement of chromosomal proteins can be analyzed in a single rapid chromatographic step.
Animals↗
The Ca2+-binding sequence in bovine brain S100b protein beta-subunit. A spectroscopic study.
Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.
Animals↗
Differential scanning calorimetry of nuclei as a test for the effects of anticancer drugs on human chromatin.
Differential scanning microcalorimetry of nuclei from cultured cells revealed differences between antitumor drugs in potency and mechanism. Scanning calorimetry of nuclei showed four structural transitions as the temperature was raised from 25 to 120 degrees C. Transitions II (76 degrees C), III (88 degrees C), and IV (105 degrees C), respectively, characterize the denaturation of the nucleosome, the unstacking of bases in nicked DNA after release from the nucleosome and unstacking in the released intact, supercoiled DNA. Nuclei from human epithelial cells treated with the DNA strand breakers bleomycin and streptonigrin showed an increase in transition III at the expense of transition IV. The effect was dose dependent. At intermediate times of treatment a substantial portion of the chromatin melted between the temperatures of transitions III and IV and this was taken to represent intact supercoiled DNA in which base-pairing had been weakened by loss of some bases. Treatment of cells with the alkylating agents N-nitroso-N-methylurea and mitomycin C gave results similar to those of the strand-breaker antitumor drugs, except that they were less potent. Irradiation by UV gave similar effects. The effects of intercalating drugs were quite distinct from those produced by strand breakers or alkylators. Nuclei from cells incubated with increasing doses of actinomycin D or ethidium bromide gave calorimetric scans that indicated progressive denaturation of the nucleosome and a concomitant stabilization of supercoiled DNA. The loss of transition IV during drug treatment was correlated with the loss of cellular capacity to divide, regardless of which drugs were used.
Alkylating Agents↗
Chromatin aggregation depends on the anion species of the salts.
The effects of anions on chromatin aggregation may be classified into three categories. First, monovalent anions, glutamate, acetate, chloride, and thiocyante, follow the lyotropic series in their effects on both H1 histone displacement and chromatin aggregation. Second, alkyl carboxylates and dicarboxylates differ in their ability to induce chromatin aggregation depending on charge density, suggesting possible interference by bulky alkyl chains with neutralization (screening) of closely spaced positive protein charges. Third, the multivalent anions, citrate3- and SO4(2-), bind tightly to histone and disrupt nucleosomes and thus interfere with chromatin aggregation. Substantial differences in chromatin aggregation were observed with different species of anions. At salt concentrations of 0-500 mN and pH 7.0, as much as 70% of the chromatin could be induced to aggregate by monosodium glutamate and sodium acetate, whereas only 10% or less was precipitated by NaSCN, Na2SO4, and Na3citrate. The physiological anion composition of the nucleus is not known; however, the anion effects discussed in the present work suggest a potential for regulation of chromatin condensation in higher eukaryotes.
Acetates↗
Chromatin aggregation changes substantially as pH varies within the physiological range.
In vitro at least, changes of pH within the physiological range are important in regulating chromatin aggregation. The extent of aggregation was shown to depend substantially on pH as well as on salt. In the absence of salt and in all of the salt conditions tested as the pH was increased, less and less chromatin was aggregated. The aggregation of half the chromatin in the presence of 0 mM NaCl, 150 mM NaCl, 150 mM NaCl and 1 mM MgCl2, and 2 mM MgCl2 was observed at pH 5.4, 6.0, 7.0, and 8.3, respectively. In 150 mM NaCl, 1 mM MgCl2, chromatin aggregation decreased from 86 to 63% as the pH was changed from 5.9 to 6.8, which is the same pH change reported to occur between interphase and mitosis. H1 dissociation from chromatin was also found to be pH dependent.
Chromatin↗