Search PubMed⌕ Search

Biomedical subjects

V G Allfrey

Publications and source records attributed to V G Allfrey.

At least 37 records · Page 2Linked to original sources
PubMed1985

Phase-I clinical trial of sodium cyanate in patients with advanced colorectal carcinoma.

Sodium cyanate, a drug that selectively suppresses amino acid incorporation for protein synthesis in tumor tissue, was given to patients with advanced colorectal carcinoma who had failed to conventional therapy, with the purpose of assessing a maximum tolerable oral dose. At 35 mg/kg p.o. daily, the drug had to be stopped in approximately half (4) of the patients because of gastrointestinal toxicity (nausea, vomiting) and neurologic toxicity (hallucinations, disorientation). However, in 5 other patients, at the same dose, the drug was well tolerated for up to 147 days and for a total cumulative dose of 308 g. In this group of patients, sodium cyanate was stopped because of evidence of tumor progression. No hematologic toxicity was observed. We observed no therapeutic effects. We therefore recommend a starting dose of 30 mg/kg p.o. if a phase-II study is considered.

Adult↗
PubMed1984

Protamine-DNA association in mammalian spermatozoa.

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.

Animals↗
PubMed1984

Microinjection studies of protein transit across the nuclear envelope of human cells.

Proteins of various molecular weights were conjugated with rhodamine and microinjected into the cytoplasm or nucleus of HeLa cells. The injected proteins were then localized within the cells at various times thereafter with fluorescence microscopy. Proteins below approx. 60 kD rapidly crossed the HeLa nuclear envelope. Some larger proteins also were able to pass into or out of the nucleus, while others were unable to do so, indicating the selective permeability of the HeLa nuclear envelope to large proteins. The nuclear protein HMG17 accumulated within the nucleus shortly after cytoplasmic microinjection.

Biological Transport↗
PubMed1983

Selective isolation of polypeptide chains bearing multiple types of postsynthetic modifications. Recovery of simultaneously acetylated and phosphorylated forms of histone H2A and high mobility group proteins 14 and 17.

In order to study coordinate or simultaneous modifications of chromosomal proteins by phosphorylation and acetylation, duck erythrocytes were incubated with [32P]orthophosphate and the thiol-containing acetate analogue, 2-mercaptoacetate. Enzymatic transfer of the analogue to the epsilon-amino groups of lysine residues permits the selective recovery of the newly thio-derivatized polypeptide chains by Hg-affinity chromatography, and this acetylated subpopulation can then be analyzed for [32P]phosphate uptake. The histones and high mobility group proteins were extracted from cell nuclei, purified, and finally analyzed for incorporation of [32P]phosphate and 2-mercaptoacetate. Several of the nuclear proteins, in particular histone H2A and the high mobility group proteins HMG-14 and HMG-17, were subjected to organomercurial-agarose chromatography. Significant amounts of these cysteine-free proteins were retained on the affinity column, and by this criterion were shown to have incorporated mercaptoacetate. The mercaptoacetylated proteins were further analyzed and found to contain the 32P label as well. These observations provide incontrovertible evidence that individual molecules of chromosomal proteins can carry postsynthetic modifications in the form of phosphorylation and acetylation at the same time, and also establish that both types of modification must have occurred during the short period in which the cells were exposed to the two precursors.

Acetylation↗
PubMed1983

Reversible changes in nucleosome structure and histone H3 accessibility in transcriptionally active and inactive states of rDNA chromatin.

The sulfhydryl reagent iodoacetamidofluorescein (IAF) was used to probe the structure of chromatin subunits in transcribed and nontranscribed regions of Physarum rDNA. IAF labels histone H3 -SH groups in the elongated monomeric subunits (A particles) from the transcribed region, but it does not label H3 in the 11S monomers from the nontranscribed central spacer. All H3 reactivity is lost from rDNA chromatin in the inactive spherule stage of Physarum. Restriction cleavage of rDNA chromatin generates fragments from the transcription unit with reactive H3 -SH groups, whereas fragments containing nontranscribed spacer sequences are unreactive. The extended rDNA chromatin contains all four core histones and other prominent proteins. Electron microscopy shows that most of the extended subunits consist of two roughly spherical bodies connected by a 50 bp nucleoprotein bridge.

Chromatin↗
PubMed1982

In vitro mercaptoacetylation of chromosomal proteins. Selective recovery of newly modified protein molecules.

Duck erythrocytes were incubated with the thiol-containing acetate analog 2-mercaptoacetate. The high mobility group proteins and histones were then extracted from the cell nuclei and the resultant protein mixtures subsequently were fractionated to their individual components by established procedures of selective trichloracetic acid precipitation and ion exchange and gel exclusion chromatography. The protein mixtures and the proteins derived from them were then subjected to mercury-affinity chromatography on organomercurial agarose columns, and it was found that significant amounts of all of the high mobility group proteins and core histones were bound by the columns. Furthermore, chymotryptic digestion of histone H4 from mercaptoacetate-treated cells and subsequent mercury-affinity chromatography revealed that the only peptides which bound to the column were the NH2-terminal peptides, the ones containing the lysines known to be the normal sites of in vivo acetylation. Carboxymethylation of mercaptoacetate-treated H4 with iodo[3H) acetate acid yielded 3H-labeled H4 and subsequent chymotryptic digestion showed that the only radioactive peptides were again those containing the lysines known to be the normal sites of enzymatic acetylation. These results and other biochemical evidence strongly indicate that the proteins of the histone and high mobility group classes which are known to be subject to postsynthetic acetyl transfer reactions can undergo postsynthetic mercaptoacetylation in vivo and that the sites of such modification are the same as those for normal acetylation. The phenomenon of mercaptoacetylation of chromosomal proteins affords a means for the selective recovery of newly modified protein molecules and their associated chromatin.

Acylation↗
PubMed1982

In vivo thiophosphorylation of chromosomal proteins. Recovery and analysis of HeLa histones and derivative phosphopeptides.

HeLa cells are shown to incorporate [35S]thiophosphate (added to the medium as sodium thiophosphate) into the gamma-positions of ATP and GTP, and into other nucleotides. Under these conditions there is a transfer of radioactive thiophosphoryl groups to histones H1, H2A, H3, and H4. The newly thiophosphorylated chromosomal proteins can be recovered selectively by affinity chromatography on organomercurial-Sepharose columns. The thiophosphorylated histone H1 and its NH2-terminal and COOH-terminal fragments were subjected to tryptic digestion and the sulfur-derivatized phosphopeptides were isolated by Hg-affinity chromatography prior to electrophoretic separation of different sites of modification. Thiophosphate was found to be incorporated into both serine and threonine residues of histone H1 in HeLa cells during logarithmic growth. When [3H]threonine and nonradioactive thiophosphate were employed simultaneously as precursors, the thiophosphorylated H1 molecules retained on the mercury column also showed the presence of the [3H]threonine label. It follows that newly synthesized H1 molecules are subject to thiophosphorylation in the growing cell cultures.

Adenosine Triphosphate↗
PubMed1982

Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.

African green monkey CV-1 cells infected with simian virus 40 (SV40) were incubated in the presence of sodium [35S]thiophosphate. The viral DNA was prepared and the newly synthesized SV40 DNA molecules containing phosphorothioate groups were selectively recovered by affinity chromatography on organomercurial-Sepharose columns. The thio-derivatized viral DNA occurred in both superhelical (form I) and relaxed circular (form II) configurations, and it gave the expected cleavage products upon digestion with the restriction endonucleases Kpn I and HindIII. Enzymatic digestion of 35S-labeled SV40 DNA to its component nucleotides and chromatographic separation of the resultant 5'-labeled phosphorothioate esters established the presence of the label in all four major nucleotides, but mainly in thymidylic acid. The ability to label and recovery selectively the newly synthesized polynucleotides by incubating cells with inorganic thiophosphate complements earlier findings [Sun, I. Y.-C. & Allfrey, V. G. (1982) J. Biol. Chem, 257, 1347--1353] that thiophosphate enters intracellular nucleotide pools and serves as an effective precursor for the phosphorylation of nuclear proteins in vivo.

Base Sequence↗
PubMed1982

Translational regulation of histone synthesis in the sea urchin strongylocentrotus purpuratus.

The pattern and schedule of histone synthesis in unfertilized eggs and early embryos of the sea urchin Strongylocentrotus purpuratus were studied using two-dimensional gel electrophoresis. After fertilization there is an abrupt change in the pattern of histone variant synthesis. Although both cleavage-stage (CS) variants. However, after fertilization, both CS and alpha messages are translated. Since alpha histone mRNA isolated from unfertilized eggs can be translated in vitro, the synthesis of alpha histone subtypes appears to be under translational control. Although the synthesis of alpha subtypes is shown here to occur before the second S phase after fertilization, little or no alpha histone is incorporated into chromatin at this time. Thus, early chromatin is composed predominantly of CS variants probably recruited for the most part from the large pool of CS histones stored in the unfertilized egg.

Animals↗
PubMed1982

Mechanisms of displacement of sperm basic nuclear proteins in mammals. An in vitro simulation of post-fertilization results.

A standardized cytological preparation of mature mouse sperm has been devised to serve as an in vitro system for probing the intra-ooplasmic molecular events of transformation of the fertilizing sperm. Two parameters of the early phase of transformation in vivo are defined at the resolution of the light microscope: deletion of sperm-unique nuclear proteins, detectable by immunofluorescence, and retention of homogeneity of the residual DNA complex, with intact chromatin boundaries detectable by ethidium bromide staining. These studies show that both parameters are conserved when in vitro sperm preparations are treated with NaCl under reducing conditions. The deletion of 2 different classes of the unique basic proteins of mouse sperm nuclei is specified by the NaCl concentration: 0.7 M-NaCl displaces the non-protamine class but not the protamines, while 1 M-NaCl displaces both. On the other hand the effects of treatment with trypsin at various concentrations and intervals are less consistent with the in vivo parameters, indicating fragmentation and displacement, not only of the sperm-unique basic proteins, but also of structural proteins believed to maintain the fundamental cohesive organization of the DNA matrix. These observations suggest that mechanisms other than proteolysis, e.g. localized changes in ionic concentrations, may participate in the post-fertilization displacement of the sperm-unique nuclear proteins in vivo. This study also supports the validity of the in vitro simulation as a model with which to probe the progression of transformation of the sperm nucleus to the zygote pronucleus.

Animals↗
PubMed1982

Aberrant and nonrandom methylation of chromosomal DNA-binding proteins of colonic epithelial cells by 1,2-dimethylhydrazine.

The alkylating carcinogen 1,2-dimethylhydrazine (DMH) induces colonic tumors with high incidence. The sites of in vivo modification of target macromolecules were studied using [methyl-3H]DMH. Carcinogen binding to subcellular fractions of colonic epithelial cells was analyzed at time intervals ranging from 10 min to 36 hr, with particular emphasis on the alkylation of nuclear constituents. DMH modifies not only nucleic acids but also histones and other DNA-binding proteins in the target cells. Separation of the 3H-methylated amino acids showed aberrant methylation patterns after exposure to [3H]DMH, as compared with methylations observed with [methyl-3H]methionine as a natural methyl group donor. DMH-modified histone H1 contained methylated lysin, arginine, and histidine residues not normally found, and other histones had abnormal methylarginine contents. High-mobility-group proteins and other nuclear proteins contained methyllysine residues not normally detected. Proteins known to be associated with template-active and more accessible DNA sequences, such as the high-mobility group proteins and the multiacetylated forms of histones H3 and H4, were preferentially damaged after exposure to [3H]DmH. The result suggests that carcinogen-induced chromosomal damage is not random and may selectively affect proteins in the actively transcribing or replicating genes in the target cells. That damage affects the amino acids most likely to be involved in DNA binding.

1,2-Dimethylhydrazine↗
PubMed1981

Manifold effects of sodium butyrate on nuclear function. Selective and reversible inhibition of phosphorylation of histones H1 and H2A and impaired methylation of lysine and arginine residues in nuclear protein fractions.

In addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.5 to 15 mM causes a selective inhibition of [32P]phosphate incorporation into histones H1 and H2A of cultured HeLa S3 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate-treated cells, but phosphorylation patterns are altered and 32P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 mM. The effect is time-dependent and fully reversible. Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of 32P-labeled histone H1 by endogenous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in vitro activities of either type I or type II cyclic AMP-dependent protein kinases, or the cAMP-independent H1 kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities. The poly(ADP)-ribosylation of HeLa histones is not inhibited by 5 mM Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.

Arginine↗
PubMed1981

Studies of acetylation and deacetylation in high mobility group proteins. Identification of the sites of acetylation in high mobility group proteins 14 and 17.

Duck erythrocytes were incubated with [3H]acetate both in the presence and absence of sodium butyrate. Subsequent perchloric acid extraction of the nuclei, followed by selective acetone precipitation, CM-Sephadex ion exchange chromatography, and gel filtration yielded radioactively labeled high mobility group (HMG) proteins HMG-14 and HMG-17 in pure form. Extensive enzymatic degradation of the proteins followed by amino acid analysis of the digests yielded a significant amount of material eluting in the position of epsilon-N-acetyllysine. Furthermore, automated Edman degradation of intact 3H-labeled HMG-14 and HMG-17 identified the specific sites of acetylation of these proteins. In both erythrocyte HMGs isolated from cells not exposed to butyrate, the lysine residue at position 2 was the only one found to be labeled. However, one additional site in HMG-14 and two additional sites in HMG-17 were found in the proteins from cells incubated in butyrate. Finally, studies of the enzymatic deacetylation of HMG-14 and HMG-17 confirmed that both nuclear proteins serve as deacetylase substrates and that butyrate inhibits their deacetylation, just as in the case of other HMG proteins and nucleosomal core histones.

Acetates↗
PubMed1981

Turnover of basic chromosomal proteins in fertilized eggs: a cytoimmunochemical study of events in vivo.

The chromosomal complements of mouse oocytes, ova, and fertilizing sperm have been studied by immunofluorescence with specific antisera to the basic protein fraction of sperm nuclei and to histones H2b and H4, and by staining with ethidium bromide. These studies support the hypothesis, previously proposed (Rodman and Barth, 1979, Dev. Biol. 68:82-95), that the chromosomes of the oocyte in maturation incorporate unique basic protein(s) similar to those incorporated during spermiogenesis. That similarity is characterized, here, by immunologic cross-reactivity. The basic proteins of the fertilizing sperm nucleus and the cross-reactive moiety of the two haploid complements of the ovum are displaced simultaneously, shortly after sperm entry. However, because the unique basic proteins incorporated into the oocyte chromosomes do not, as in the spermatogenic sequence, entirely replace the histones, the maternal chromosomes display histones H2b and H4 at all postfertilization stages examined, whereas the decondensing paternal complement, for an interval before maturation of the pronuclei, contains neither sperm basic chromosomal proteins nor histones. Sequential staining of the same specimens with ethidium bromide revealed well-organized nuclear morphology of the residual DNA complex. Those observations suggest that, for an as yet undefined period in the transformation from spermatozoal to embryonic genome, the chromatin is devoid of a complement of basic proteins.

Animals↗
PubMed1981

Activation of sodium cyanate for selective inhibition of protein synthesis in cultured tumor cells.

Sodium cyanate, a selective inhibitor of protein synthesis in animal tumors in situ, has no comparable effect when added to tumor cells in culture. However, a rapid inhibition of protein synthesis in cultured tumor cells can be achieved by reaction of cyanate with the drug-metabolizing system of liver microsomes. Procedures are described for the induction, preparation, and testing of an active cytochrome P-450 fraction which converts cyanate to a short-lived dialyzable metabolite that selectively inhibits amino acid incorporation in tumor cells without a corresponding effect on protein synthesis in normal cells. The suppression of tumor protein synthesis is not due to a general toxicity reaction because thymidine incorporation into the DNA of tumor cells is not inhibited by the cyanate metabolite. When HeLa cells are exposed to sodium butyrate, a substance reported to suppress the malignant phenotype in several tumor cell lines, they lose their sensitivity to the cyanate metabolite. Chick fibroblasts which are normally insensitive to the cyanate metabolite become cyanate sensitive after transformation by the Schmidt-Ruppin strain of the Rous sarcoma virus.

Adenocarcinoma↗