Biomedical subjects
Publications and source records attributed to R C Miller.
Hybrid cells obtained from A-T and D98/AH(HeLa) cells showed normal radiation sensitivity to cell killing, but retained radioresistant DNA synthesis similar to parental A-T cells.
1. The aim of this study was to examine the possible role of the release of guanosine 3':5'-cyclic monophosphate (cyclic GMP) into the extracellular space in the regulation of rat aortic cyclic GMP content. 2. Rat aortic segments incubated in physiological solution released cyclic GMP into the medium in a time-dependent manner. This release was greatly enhanced when intact instead of tissues without endothelium were used. After 120 min of observation, a maximal 33 fold difference in extracellular cyclic GMP content was detected. 3. Treatment of rat aortic preparations with either a Ca2+-free solution or methylene blue, both conditions known to inhibit endothelium-derived relaxing factor (EDRF)-mediated responses, markedly reduced the extracellular accumulation of cyclic GMP from tissues with but not without endothelium. 4. Endothelium-dependent vasodilators such as acetylcholine (10 microM) and carbachol (10 microM) greatly increased tissue cyclic GMP content, in a time-dependent manner in rat aortic preparations with endothelium, but only slightly in tissues without. Maximal increases in intact tissues were obtained after about 1 min of agonist contact and amounted to about 35 and 15 fold respectively, thereafter tissue cyclic GMP content rapidly declined. Histamine (10 microM) elicited only minor effects on tissue cyclic GMP content of both intact preparations and those without endothelium. 5. Acetylcholine (10 microM), carbachol (10 microM) and histamine (10 microM) stimulated a time-dependent release of the cyclic nucleotide into the incubation medium from tissues with endothelium. After 120 min of observation, extracellular accumulation of cyclic GMP from intact tissues was increased by about 2.6, 6.6 and 1.7 fold respectively. Carbachol and histamine induced only minor effects on release from tissues without endothelium. 6. Sodium nitroprusside (0.3 and 10 microM), a direct activator of soluble guanylate cyclase, induced a concentration-dependent accumulation of cyclic GMP in tissues with and without endothelium that was associated with a concentration-dependent accumulation of cyclic GMP in the extracellular space. Peak tissue cyclic GMP content reached similar levels in preparations with and without endothelium, while extracellular cyclic GMP levels were about two times greater when experiments were performed with intact compared to endothelium-denuded tissues. 7. Atriopeptin II, an activator of particulate guanylate cyclase, increased tissue cyclic GMP content by about 8 and 18 fold respectively in tissues with and without endothelium. As was the case with sodium nitroprusside, atriopeptin II-stimulated release was markedly enhanced from intact tissues compared with those without endothelium. After 120 min of observation, there was a 16 fold difference in the amount of extracellular cyclic GMP.
1. The aim of the present study was to examine the contractile responses evoked by the recently characterized vasoactive peptide endothelin-1 (ET-1) and by two of its structural analogues, [Ala3,11]ET-1 and [Ala1,15]ET-1 in endothelium-denuded rat isolated aorta, and also to assess the extent of dependence of these responses on extracellular calcium entry. 2. ET-1 (0.3 to 10 nM), [Ala3,11]ET-1 (2.25 to 225 nM) and [Ala1,15]ET-1 (0.04 to 1.36 microM) evoked concentration-dependent contractions in normal, calcium-containing medium with the order of potency: ET-1 greater than [Ala3,11]ET-1 greater than [Ala1,15]ET-1. 3. Preincubation of tissues for 60 min with diltiazem (1 microM) induced a significant 3 fold rightward shift of concentration-effect curves to ET-1 without affecting maximal responses elicited by 10 nM of this peptide, whereas the same treatment failed to modify concentration-effect curves to [Ala3,11]ET-1. 4. Preincubation of tissues for 60 min with nifedipine (0.1 or 1 microM) markedly inhibited contractions elicited by either ET-1 (10 nM) or [Ala1,15]ET-1 (0.41 microM). Furthermore, when added cumulatively to tissues maximally contracted by ET-1 (10 nM), nifedipine (3 nM to 1 microM) induced concentration-dependent relaxations with an IC50 value of 21.8 +/- 5.9 nM. Maximal relaxation to nifedipine, 1 microM, amounted to 56.9 +/- 11.5%. 5. Submaximal concentrations of ET-1 (3 nM), [Ala3,11]ET-1 (75 nM) and [Ala1,15]ET-1 (0.41 microM), gave about 85% of maximal contractions elicited by noradrenaline (1 microM) in normal, calcium-containing medium. These contractile responses were all reduced by about 70% in calcium-free medium. These contractile responses were all reduced by about 70% in calcium-free medium. Pretreatment of tissues with diltiazem (1 microM) or with nifedipine (0.1 or 1 microM) did not affect these residual contractions. 6. Upon readdition of calcium (10 microM to 10mM) to tissues in calcium-free medium, preincubated with submaximal concentrations of one or the other of the peptides, concentration-dependent contractions were elicited with EC50 values of 1.21 + 1.1 mm (ET-1-exposed rings), 74.6 + 9.1 microM ([Ala31 ']ET-1-exposed rings) and 102 + 27 microM ([Ala' 15]ET-1-exposed rings). Calcium-induced responses were significantly inhibited by diltiazem (1 microM) in both ET-1- and [Ala3"1 ']ET-1-exposed tissues and by nifedipine (0.1 and 1 microM) in ET-1-exposed tissues. However, nifedipine did not significantly affect calcium induced responses in [Ala"'15]ET-1-exposed rings. 7. Readdition of calcium to a calcium-free medium containing 40mm K+, evoked concentrationdependent responses that were unaffected by the presence of ET-1 (3 nM). 8. Overall, these results indicate that contractions to ET-1 and its analogues in rat aorta are to some extent dependent on extracellular calcium. However, the mode of antagonism by diltiazem and nifedipine of responses to the peptides in normal medium, as well as of contractions induced by readdition of calcium in peptide-exposed tissues in calcium-free medium, argue against a direct activation by endothelin or its analogues of L-type calcium channels. Finally, removal of one or the other of the disulphide bonds of ET-1 reduces the potency of the peptide.
Two nonglycosylated endoglucanases which bind to Sephadex were purified from culture supernatants of Cellulomonas fimi grown on microcrystalline cellulose. Their Mrs were 120,000 and 130,000. The N-terminal amino acid sequences of the enzymes were identical, suggesting that the enzymes were related. A DNA fragment encoding this N-terminal sequence was cloned in Escherichia coli. The nucleotide sequence corresponding to the N-terminal amino acid sequence was preceded by a sequence encoding a typical leader peptide. Transcripts hybridizing to the cloned fragment were detected in total RNA isolated from C. fimi cells grown on carboxymethyl cellulose but not from cells grown on glycerol or glucose. Transcription started at a cluster of sites 53 to 59 nucleotides upstream of a GUG translation initiation codon and terminated at either of two closely spaced C residues immediately downstream of a region of potential secondary structure. The size of the transcript was approximately 3.5 kilobases, sufficient to encode a polypeptide of 130 kilodaltons. The 130-kilodalton polypeptide is designated endoglucanase C (CenC), and the gene encoding it is designated cenC.
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Cultured human thyroid cells were X-irradiated in vitro and assayed for resistance to 6-thioguanine. The average mutant frequency was 1.69 +/- 1.34 X 10(-5) (mean +/- SD) in controls, 3.74 +/- 2.21 X 10(-5) in cells exposed to 1 Gy, and 7.19 +/- 5.37 X 10(-5) in cells exposed to 2 Gy. The positive association between mutant frequency and dose was statistically significant. The estimated mutation induction rate was 2.54 +/- 0.71 X 10(-5) per gray, which is in close agreement with published results for human skin fibroblasts and mammary epithelial cells. These results extend and confirm earlier reports that mutation induction rates for fibroblasts and epithelial cells after exposure to X rays are similar.
After exposure to various doses of 250 kVp X radiation, 0.85 Me V fission spectrum neutrons, or 600 MeV/A iron (Fe) particles, mitotically quiescent rat lens cells showed no visible evidence of radiation injury. However, following the mitogenic stimulus of wounding, mitotic abnormalities became evident when responding cells entered mitosis. Latent damage and recovery therefrom were monitored at 3, 7, 14, and 28 days after irradiation. Following doses of 1 to 10 Gy of X radiation, the recovery rate, indicated by a decrease in abnormalities with time, was proportional to dose, and the dose-effect slope decreased exponentially with time. Virtually no recovery occurred during the 28 days after 1.25 to 2.25 Gy of fission neutron radiation. After doses of 0.5 to 3.0 Gy of Fe particles, an increased expression of mitotic damage or recovery than recovery occurred. As a consequence of the differing patterns in time for expression of damage or recovery following X rays and the high-LET radiations, the relative biological effectiveness (RBE) increased from 3.6 to 16 for neutrons and from 2 to 10 for Fe particles over the 28-day observation period.
The relative biological effectiveness (RBE) of a range of neutron energies relative to 250-kVp X rays has been determined for oncogenic transformation and cell survival in the mouse C3H 10T 1/2 cell line. Monoenergetic neutrons at 0.23, 0.35, 0.45, 0.70, 0.96, 1.96, 5.90, and 13.7 MeV were generated at the Radiological Research Accelerator Facility of the Radiological Research Laboratories, Columbia University, and were used to irradiate asynchronous cells at low absorbed doses from 0.05 to 1.47 Gy. X irradiations covered the range 0.5 to 8 Gy. Over the more than 2-year period of this study, the 31 experiments provided comprehensive information, indicating minimal variability in control material, assuring the validity of comparisons over time. For both survival and transformation, a curvilinear dose response for X rays was contrasted with linear or nearly linear dose responses for the various neutron energies. RBE increased as dose decreased for both end points. Maximal RBE values for transformation ranged from 13 for cells exposed to 5.9-MeV neutrons to 35 for 0.35-MeV neutrons. This study clearly shows that over the range of neutron energies typically seen by nuclear power plant workers and individuals exposed to the atomic bombs in Japan, a wide range of RBE values needs to be considered when evaluating the neutron component of the effective dose. These results are in concordance with the recent proposals in ICRU 40 both to change upward and to vary the quality factor for neutron irradiations.
An endo-beta-1,4-glucanase (CenA) and an exo-beta-1,4-glucanase (Cex) were prepared from Escherichia coli expressing recombinant DNA of the cellulolytic bacterium Cellulomonas fimi. Purification was facilitated by the high affinities of these enzymes for cellulose. An extracellular C. fimi protease cleaved both enzymes in vivo in a highly specific manner. The affinity of the parent enzyme for cellulose was contained independently in an amino-terminal fragment (p20) of CenA and a carboxyl-terminal fragment (p8) of Cex. These fragments contained homologous amino acid sequences which were proposed to comprise cellulose binding domains. Corresponding fragments, p30 from CenA and p35 from Cex, which were unable to bind to cellulose, contained catalytic domains. In both enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from CenA by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge. Cex was cleaved at an exactly analogous site. p30 and p35 retained several of the catalytic functions of their parent enzymes. However, p30 was less active than intact CenA against microcrystalline cellulose implying a critical role for the binding domain of CenA in the hydrolysis of crystalline substrate.
The modulating effect of 43 degrees C hyperthermia on the induction of oncogenic transformation by the antineoplastic agents, actinomycin D, mitomycin C, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was examined using the C3H 10T1/2 cell line. For any given level of cytotoxicity, cells exposed to the three chemotherapy agents at 37 degrees C showed similar frequencies of transformation. Transformation frequencies induced by all three drugs were reduced by hyperthermia. The reduction was most pronounced for cells exposed to BCNU, and to a lesser extent, by cells exposed to actinomycin D and mitomycin C. The modulating effects of heat on drug-induced transformation incidence appeared to be independent of whether application of heat and drug was concurrent or sequential.
Dosimetry and biological effects of 40 and 50 keV low-energy X-rays generated by a SOFTEX Model CMBW-2 apparatus were studied. Doses were measured using a thin-window parallel-plate ionization chamber; beam quality was assessed using aluminum absorbers; exposure rates per unit current were determined according to the X-ray tube current and exposure times; and thermoluminescent (BeO chip) dosimeters were used to ascertain dose distributions in the irradiation field. The average correction factors for nonuniformity were calculated from the measured dose distributions. The means for ascertaining accurate exposures and doses using these methods are discussed. The dose-survival relationship of Chinese hamster V79 cells were assessed by irradiating them with 40 and 50 kV soft X-rays, 180 kV X-rays, and 60 Co gamma rays. Soft X-rays with three distinct effective energies were tested by changing the tube voltage kV and aluminium filter thicknesses; namely (1) 40 kV without filter, (2) 40 kV with a 0.2 mm thick aluminium filter and (3) 50 kV with a 0.7 mm thick aluminium filter. The effective energies obtained according to attenuation measurements using aluminium for these soft X-rays were 8.1, 11.7 and 18.5 kV, respectively. In this study the relative biological effectiveness (RBE) at 10 per cent survival compared with 60Co gamma rays ranged from 1.5 to 1.6. The RBE of 180 kV X-rays relative to 60Co gamma rays was 1.29. This study provided experimental data for the RBE of V79 cells in the intermediate energy range between hard and ultrasoft X-rays, data for which were previously reported by Goodhead and co-workers (1977, 1979, 1981).
We used the yeast MEL1 gene for secreted alpha-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi beta-1,4-exoglucanase was inserted into one cartridge to create a fusion of the alpha-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-beta-d-cellobiose, and p-nitrophenyl-beta-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical K(m) and pH optimum but to be more thermostable.
Endoglucanase B (EB) of Cellulomonas fimi has an M(r) of 110,000 when it is produced in Escherichia coli. The level of expression of the cenB gene (encoding EB) was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. EB was purified to homogeneity from the periplasmic fraction of E. coli in one step by affinity chromatography on microcrystalline cellulose (Avicel). Alignment of the NH(2)-terminal amino acid sequence with the partial nucleotide sequence of a fragment of C. fimi DNA showed that EB is preceded by a putative signal polypeptide of 33 amino acids. The signal peptide functions and is processed correctly in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of beta-galactosidase. The intact EB polypeptide is not required for enzymatic activity. Active polypeptides with M(r)s of 95,000 and 82,000 also appear in E. coli, and a deletion mutant of cenB encodes an active polypeptide with an M(r) of 72,000.
The DNA sequence was determined for the cloned Agrobacterium sp. strain ATCC 21400 beta-glucosidase gene, abg. High-resolution nuclease S1 protection studies were used to map the abg mRNA 5' and 3' termini. A putative abg promoter was identified whose sequence shows similarities to the consensus promoter of Escherichia coli and with the nif promoter regions of Klebsiella. The abg coding sequence was 1,374 nucleotides long. The molecular weight of the enzyme, based on the predicted amino acid sequence, was 51,000. The observed Mr was 50,000 to 52,000. A region of deduced protein sequence was homologous to a region from two other beta-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme.
Quiescent cells of the central zone region of the rat lens epithelium were stimulated to enter the proliferation cycle by wounding. RNA synthesis and a corresponding increase in poly(A)+/total RNA reached a peak by Hour 4. Cells progressed into the G1B compartment by Hour 10. A rise in protein synthesis began at Hour 8, and onset of DNA synthesis occurred by Hour 14. The timing of cell cycle progression that allowed escape from a dose of X irradiation that completely inhibited DNA synthesis was investigated. A growth-arrest point was identified at Hour 9 where 10 GY of X irradiation given before, but not after, completely inhibited earliest responding cells from entering DNA synthesis on schedule. Increased quantities of cells entered DNA synthesis on schedule as timing of the X irradiation was moved closer to the end of G1. Based on time relationships, the rise in protein synthesis is correlated with the "sufficient" event for the escape.
Primary, first and second passaged endothelial cells from bovine aorta were grown in plastic culture dishes or on glass coverslips. The cells were characterized by their monolayer cobblestone appearance at confluence, their immunofluorescent staining for factor VIII-related antigen, their specific uptake of low density lipoprotein and by their ultrastructure. Following stimulation of the cells by atriopeptin II or sodium nitroprusside, both cellular and extracellular cyclic GMP levels were measured. Cellular cyclic GMP content was increased greatly by atriopeptin II in a time-dependent manner while sodium nitroprusside was essentially without effect. Increases in tissue cyclic GMP levels were associated with a time-dependent accumulation of the nucleotide in the extracellular compartment. Zaprinast, a specific inhibitor of cyclic GMP phosphodiesterases, did not significantly affect either basal or atriopeptin II-stimulated increases in cyclic GMP content, nor extracellular accumulation of the nucleotide. It is concluded that the cyclic GMP content of endothelial cells is not solely dependent on degradation by phosphodiesterases but also involves release of cyclic GMP into the extracellular compartment.
Subpopulations of mouse lens epithelial cells, differing in proliferative status, were irradiated with either X rays or fission spectrum neutrons given singly or in four weekly fractions. After various times, epithelia were mitogenically stimulated by wounding and DNA synthesis responses were determined by incorporation of [3H]thymidine. At 1 h following both X and neutron irradiations, significant suppression of the wound response after single doses and a sparing effect of fractionation were evident in both the mitotically quiescent and the slowly proliferating subpopulations. At 1 week following single or fractionated doses of both radiations, recovery was evident in both subpopulations. By 4 weeks, the quiescent subpopulation showed significant recovery after both single and fractionated doses of X rays or neutrons. In contrast, a marked decreased ability to respond after neutron irradiation and, in addition, a significant enhancement effect of neutron fractionation were observed for the slowly proliferating subpopulation. Per gray, neutrons were about 7.5 times more effective than X rays as a single dose and 25 times more effective in four equal fractions. The shift from an initial sparing to a final enhancing effect of neutron fractionation for the slowly proliferating subpopulation has importance for understanding divergent early and late radiation responses following dose fractionation.
Oncogenic transformation was assayed after C3H 10T1/2 cells were irradiated with monoenergetic neutrons; cells were exposed to 0.23-, 0.35-, 0.45-, 5.9-, and 13.7-MeV neutrons given singly or in five equal fractions over 8 h. At the biologically effective neutron energy of 0.45 MeV, enhancement of transformation was evident with some small fractionated doses (below 1 Gy). When transformation was examined as a function of neutron energy at 0.5 Gy, enhancement was seen for cells exposed to three of the five energies (0.35, 0.45, and 5.9 MeV). Enhancement was greatest for cells irradiated with 5.9-MeV neutrons. Of the neutron energies examined, 5.9-MeV neutrons had the lowest dose-averaged lineal energy and linear energy transfer. This suggests that enhancement of transformation by fractionated low doses of neutrons may be radiation-quality dependent.