Biomedical subjects
Publications and source records attributed to D A Prentice.
The occurrence of bilateral brachial mononeuropathies presenting as anterior interosseous nerve syndromes in association with cytomegalovirus mononucleosis in an otherwise healthy adult is reported, a relationship not previously described.
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Three patients who were admitted to hospital because of syncope were found to have malignant head and neck tumours. A patient who presented with malignant external otitis with syncope and postural hypotension is also described. It is postulated that the syncope and postural hypotension result from compressive irritation of the glossopharyngeal nerve either in the neck or at the base of the skull.
We have recently developed a reproducible method for the use of DNase I as a sensitive probe of chromatin structure (Prentice, D A & Gurley, L R, Biochim biophys acta 740 (1983) 134) [12] and have used this probe to investigate chromatin structure during the interphase of the cell cycle. Chinese hamster cells (line CHO) were synchronized by: (1) mitotic detachment, to obtain M-phase cells; (2) isoleucine deprivation, to obtain G1-phase cells; and (3) sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea, to obtain cells blocked at the start of S phase. The cells were released from the various blocking schemes and nuclei were isolated and digested with DNase I at various times. The digestion kinetics were monitored to detect possible changes in chromatin condensation through the cell cycle. The chromatin was much more accessible to DNase I in G1 phase than in S or G2 phase, with only small variations in structure detected in late G1 and very early S phase. From early S phase up to mitosis, the chromatin became increasingly condensed and inaccessible to DNase I action. These results support the concept of a chromatin condensation cycle during interphase as well as during mitosis.
Nucleases such as DNase I, which selectively digest chromatin, are inhibited by several commonly used phosphatase inhibitors including sodium bisulfite. Two inhibitors, sodium arsenate and fructose-1,6-diphosphate, did not significantly inhibit nuclease action. Two other effective phosphatase inhibitors, p-chloromercuriphenyl sulfonate and 5,5'-dithiobis(2-nitrobenzoate), can be used during nuclei isolation and then washed out of nuclei before nuclease digestion. Using this procedure, 1mM p-chloromercuriphenyl sulfonate is as effective as 50mM bisulfite in retaining the phosphatase-sensitive mitotic phosphorylations of histones H1 and H3.
Methods have been developed for the preparation of histone variants and high-mobility group (HMG) proteins by high-performance liquid chromatography (HPLC). The individual HPLC fractions were recovered as a dry powder in 95% yield by direct lyophilization from the column effluent. Perchloric acid-soluble H1 variants and HMG proteins from Chinese hamster cells (line CHO) were separated on a mu Bondapak CN column using a 0-50% linear acetonitrile gradient in water containing 0.2% trifluoroacetic acid (TFA). The proteins were eluted in the following order: HMG-E/G (an HMG-14/17 class proteins from CHO cells), HlO, Hl, HMG-2, and HMG-l. HMG-E/G, Hl, and an unidentified protein were recovered electrophoretically pure. HlO contained contaminants which could be removed by subsequent chromatography on a mu Bondapak C18 Radial-Pak column, but HMG-l and HMG-2 could not be completely resolved. Nucleosomal core histones were fractionated on a mu Bondapak C18 Radial-Pak column using a 30-55% linear acetonitrile gradient containing 0.2-0.3% TFA. They were eluted in the following order: H2B, (LHP)H2A, (MHP)H2A, H4, LHP(H3), and (MHP)H3, (where LHP and MHP refer to less-hydrophobic and more-hydrophobic variants). If the gradient containing 0.3% TFA was interrupted with an isocratic elution at 43% acetonitrile, the H2B, (LHP)H2A, (MHP)H2A, and H4 proteins were completely resolved, thus providing a good preparative method for these proteins. The H2A class of Drosophila histones was also fractionated on a mu Bondapak C18 Radial-Pak column using a 30-35% linear acetonitrile gradient containing 0.2% TFA. Drosophila melanogaster H2A, obtained as a single fraction by chromatography on Biol-Gel P-100, was eluted from the C18 column as three proteins. The order of elution was identified by electrophoresis to be: H2Aox (an oxidized form of H2A), D2 (a Drosophila-specific subtype), and H2A.
The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134-144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G1 phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G1 phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G1- and S-phase cells, respectively. The G1 cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G1 nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.
We have investigated the loss of histone H1 from chromatin [D'Anna, J. A., Gurley, L. R., & Tobey, R. A. (1982) Biochemistry 21, 3991-4001] and the structure of chromatin from Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation G1 block and 1 mM hydroxyurea (HU) blockade. Measurements of H1 content in the cell and histone turnover indicate that H1 is lost from the cell and that there is negligible replacement synthesis of H1 during the period of the S-phase block. As H1 is lost, chromatin appears to undergo structural change. After 10 h of HU block, the new deoxyribonucleic acid (DNA) and a portion of the old DNA have measured nucleosome repeat lengths (37 degrees C digestion) which are less than those of controls and similar to those observed by Annunziato and Seale [Annunziato, A. T., & Seale, R. L. (1982) Biochemistry 21, 5431-5438] for new immature chromatin in the absence of HU. By 24 h of HU block, nearly all of the chromatin has assumed a pseudoimmature conformation in which the nucleosome cores appear to be more closely packed along the DNA chain, but the new DNA is slightly more resistant than old DNA to attack by micrococcal nuclease. Electrophoretic analysis of nucleoprotein particles produced by micrococcal nuclease digestion of nuclei indicates that (1) the distribution of mononucleosome species changes during HU block and (2) some mononucleosome species appear to be enriched in normally minor proteins which may determine the electrophoretic mobility of the nucleoprotein particles in agarose-acrylamide gels. The results raise the possibility that (1) during the early stages of replication (or prior to the passage of the replication fork), H1 is dissociated from initiated replicons and (2) H1 does not reassociate in a concerted fashion with the H1-depleted chromatin until the replication fork has passed and, perhaps, a substantial portion of the replicon has been replicated.
A method is described for the rapid analysis of histones by high-performance liquid chromatography on reversed-phase muBondapak columns, containing either octadecylsilane (C18) or cyanopropylsilane (CN) bonded to silica particles packed in either steel columns or Radial-Pak cartridges. A linear gradient progressing from water-acetonitrile (80:20) to water-acetonitrile (40:60) and increasing in acetonitrile concentration at the rate of 10%/h was used to elute the histones at a flow-rate of 1 ml/min for steel columns or 2 ml/min for Radial-Pak cartridges. Two conditions were found to be necessary to achieve histone fractionation: (1) silylation of the active silanol groups on the silica matrix, and (2) 0.1-0.3% trifluoroacetic acid in the eluting solvent. More than 95% of the total [3H]lysine-labeled protein applied to the CN column was eluted. The histone fractions were identified by their electrophoretic mobilities in both acid-urea and Triton DF-16 polyacrylamide gels. Histones were eluted from the columns in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less-hydrophobic and more-hydrophobic histone variants). Phosphorylated and acetylated histone molecules were not separated from their unmodified parent molecules. The volatile nature of the water-acetonitrile-trifluoroacetic acid eluting solvent facilitated recovery of salt-free histones from the fractions by direct lyophilization of the column effluents. The best resolution of histone fractions was obtained with the Radial-Pak muBondapak C18 cartridge using 0.3% TFA. However, for analytical studies, the best detection was obtained by using the muBondapak CN steel column. Poorer resolution was obtained by using the non-silica based PRP-1 reversed-phase column, containing a polystyrene-divinylbenzene resin under the same conditions.
Experiments using nucleases as probes of chromatin structure take place in two stages: (1) nuclei isolation, and (2) nuclease digestion. The parameters of the nuclease digestion stage are usually strictly controlled because of nuclease sensitivity to them. However, there have been no reports on whether parameters in the nuclei isolation stage affect the subsequent nuclease digestions. We have evaluated a typical nuclei isolation technique with respect to how changes in the isolation parameters affect nuclease digestion kinetics. Our observations point out that various parameters encountered in the nuclei isolation stage have a significant effect on the subsequent nuclease digestion kinetics of DNAase I. These parameters include the concentration of cells, divalent cations and phosphatase inhibitors. The pH, concentration of NaCl and concentration of detergent had little effect. Micrococcal nuclease was relatively unaffected by changes in the nuclei isolation parameters. The importance of this report lies in the demonstration that lack of control of seemingly insignificant parameters, such as cell concentration during the nuclei isolation stage, leads to subsequent irreproducible results in the DNAase I digestion. These findings indicate that great care must be exercised in the nuclei isolation stage if reproducible work is to be performed with DNAase I.
A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase mu Bondapak C18 column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total [3H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.
Previous work in our laboratory [Gurley et al. Anal. Biochem. 129, 132-144 (1983)] described conditions for the rapid fractionation of histones by high-performance liquid chromatography (HPLC) using a reverse-phase muBondapak C18 column. That procedure resolved the major classes of histones with one exception: the more hydrophobic H2A variant, (MHP)H2A, was not resolved from the H4 histone class. This report extends that work describing experiments using a muBondapak CN column which better resolves the classes of histones from each other including the resolution of (MHP)H2A from the H4. In addition, the less hydrophobic H2A variant, (LHP)H2A, is partially resolved from the (MHP)H2A, and the less hydrophobic H3 variant, (LHP)H3, is resolved from the more hydrophobic H3 variant, (MHP)H3. Lower trifluoroacetic acid (TFA) concentrations (0.1%) in the eluting water/acetonitrile solvent were used with the CN column than were used with the C18 column which increased the sensitivity of histone detection by ultraviolet absorption at 206 nm. Greater than 95% of the total [3H]lysine-labeled protein applied to the CN column was eluted from the column. Contaminating nonhistone proteins were found to chromatograph in the region of histone elution. These were greatly reduced by isolating nuclei prior to histone preparation. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The histone fractions (identified by their electrophoretic mobilities) were eluted from the CN column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3. Phosphorylated and acetylated histone species were not resolved from their unmodified parental species.
We have sought to determine the role of histone H2A phosphorylation in chromatin by examining the distribution of the phosphorylated and unphosphorylated forms of this core histone within the nuclei of mouse and human cells. At any time, only about 15% of the H2A of whole chromatin is in the phosphorylated form, and its phosphate is rapidly turned over, even in quiescent cells that contain a functional nucleus. The phosphorylations and dephosphorylations are not specifically relate to progress through the cell cycle, nor to DNA synthesis or repair, and they are not selectively nucleolar. Euchromatin is substantially enriched with phosphorylated H2A but is not the exclusive repository of it. Possible roles of this modification of H2A are considered.
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Rat follicle stimulating hormone (rFSH) was analysed in extracts of rat anterior pituitary using a newly developed separation method of reversed-phase high performance liquid chromatography (RP-HPLC) and post-column detection of immunoreactivity (rFSH-IR) by radioimmunoassay (RIA) or immunoprecipitation with a specific anti-rFSH antibody. Good separation of rFSH was achieved on a Waters Radial-Peak 8MBC18 cartridge with a linear (60 min) gradient from 10-60% acetonitrile at constant 0.1% trifluoroacetic acid concentration. The elution profiles of radioiodinated purified rFSH [(125I)rFSH] as well as extracts of adult male and female rat pituitaries contained two main immunoreactive components (peaks 1 and 2) which eluted at 16-17 min and 21 min respectively. In the (125I)rFSH elution profile, the peaks of immunobinding corresponded to two major peaks of radioactivity. The male pituitary showed greater binding to anti-rFSH-serum of peak 1 and 2 eluate compared to female pituitary. At day 20 after orchidectomy, the amount of immunoreactive rFSH (rFSH-IR) increased in both peaks (by 30% in peak 1 and 180% in peak 2); however, the rFSH-IR level in peak 2 appeared to be more variable compared with that in peak 1. Subcutaneous implantation of 5 mg or 15 mg of dihydrotestosterone (DHT) at the time of orchidectomy prevented the post-castration elevation of rFSH-IR at 10 and 20 days after surgery. The precise nature and physiological significance of the two elution peaks are not clear at this time; however, only peak 2 showed immunoreactivity with anti-rLH-alpha-subunit antibody (AFP 87713681) and the elution time of peak 2 coincided with that of (125I) rLH-alpha subunit (AFP-7264 B).(ABSTRACT TRUNCATED AT 250 WORDS)