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Inhibition of cell division by beryllium (Be2+) has been examined in synchronized cultures of a liver-derived cell line (BL9L cells) using cytofluorometric cell cycle analysis. Results show that a selective dose-related block of the G1-pre-S transition is produced, with other periods of the cell cycle appearing relatively insensitive.
Stable inactive 2 : 1 complexes of the Klebsiella pneumoniae nitrogenase components (Kp2/Kp1) were prepared with ADP or the fluorescent ADP analogue, 2'(3')-O-[N-methylanthraniloyl] ADP and AlF(4)(-) or BeF(3)(-) ions. By analogy with published crystallographic data [Schindelin et al. (1997) Nature 387, 370-376)], we suggest that the metal fluoride ions replaced phosphate at the two ATP-binding sites of the iron protein, Kp2. The beryllium (BeF(x)) and aluminium (AlF(4)(-)) containing complexes are proposed to correspond to the ATP-bound state and the hydrolytic transition states, respectively, by analogy with the equivalent complexes of myosin [Fisher et al. (1995) Biochemistry 34, 8960-8972]. (31)P NMR spectroscopy showed that during the initial stages of complex formation, MgADP bound to the complexed Kp2 in a manner similar to that reported for isolated Kp2. This process was followed by a second step that caused broadening of the (31)P NMR signals and, in the case of the AlF4- complex, slow hydrolysis of some of the excess ADP to AMP and inorganic phosphate. The purified BeFx complex contained 3.8 +/- 0.1 MgADP per mol Kp1. With the AlF(4)(-) complex, MgAMP and adenosine (from MgAMP hydrolysis) replaced part of the bound MgADP although four AlF(4)(-) ions were retained, demonstrating that full occupancy by MgADP is not required for the stability of the complex. The fluorescence emission maximum of 2'(3')-O-[N-methylanthraniloyl] ADP was blue-shifted by 6-8 nm in both metal fluoride complexes and polarization was 6-9 times that of the free analogue. The fluorescence yield of bound 2'(3')-O-[N-methylanthraniloyl] ADP was enhanced by 40% in the AlF(4)(-) complex relative to the solvent but no increase in fluorescence was observed in the BeFx complex. Resonance energy transfer from conserved tyrosine residues located in proximity to the Kp2 nucleotide-binding pocket was marked in the AlF(4)(-) complex but minimal in the BeFx fluoride complex, illustrating a clear conformational difference in the Fe protein of the two complexes. Our data indicate that complex formation during the nitrogenase catalytic cycle is a multistep process involving at least four conformational states of Kp2: similar to the free Fe protein; as initially complexed with detectable (31)P NMR; as detected in mature complexes with no detectable (31)P NMR; in the AlF(4)(-) complex in which an altered tyrosine interaction permits resonance energy transfer with 2'(3')-O-[N-methylanthraniloyl] ADP.
Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization. We have determined how a decreased hydrophobicity of this plug affects yeast actin function. Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern. However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells. V266D actin polymerizes slower than wild-type actin at room temperature. At 4 degrees C, not only is polymerization slowed, but there is also an effect on critical concentration. However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu267. Purified GG-actin does not polymerize in vitro alone or in the presence of wild-type F-actin seeds. GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4 degrees C only phalloidin is effective. These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization. However, the V266D actin results require a modification of the original Holmes filament model.
The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca(2+)-ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), were explored and compared with those of actual E2P formed from P(i) without Ca(2+). Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca(2+) release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF.E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca(2+)-ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca(2+) (over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca(2+) is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca(2+) release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF(3)(-)) to trigonal bipyramidal transition state (AlF(3) or AlF(4)(-)) and further to tetrahedral product state (MgF(4)(2-)), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca(2+).
The effect of beryllium (Be) compounds on porphyrins was investigated in pregnant mice. The blood protoporphyrin (Proto) and zinc protoporphyrin (Zn Proto) concentrations were increased in pregnancy. Regardless of pregnancy or nonpregnancy, the Proto concentration was decreased after Be injection. Delta-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) activities in blood were significantly elevated in the pregnant untreated (Con-pregnant) group, compared to the nonpregnant mice untreated (Con-nonpregnant) and nonpregnant mice treated with Be (Be-nonpregnant) groups. The blood ALA-D activity of the pregnant mice treated with Be (Be-pregnant group) tended to decrease, compared to Con-pregnant group. The blood PBG-D activity in the Be-pregnant group was significantly lower compared with that of the Con-pregnant group. The ALA-D and PBG-D activities in the spleen were also significantly elevated in the Con-pregnant group, compared to nonpregnant groups. However, it was noted that these values in the Be-pregnant group were almost the same as that of the Con-nonpregnant group and were significantly lower than that in the Con-pregnant group. The elevation of ALA-D and PBG-D activities in the blood and spleen, which play a role in the hematopoietic function of mice, was observed in the Con-pregnant mice compared to the nonpregnant mice. However, the phenomenon was not observed in the Be-pregnant mice, it suggesting that Be suppressed the pregnancy-induced increase in hematopoietic function.
Natural radionuclides have been proposed as a means of assessing the transport of ozone (O3) and aerosols in the troposphere. Beryllium-7 (7Be) is produced in the upper troposphere and lower stratosphere by the interaction of cosmogenic particles with atmospheric nitrogen and oxygen. 7Be has a 53.29-day half-life (478 keV gamma) and is known to attach to fine particles in the atmosphere once it is formed. It has been suggested that O3 from aloft can be transported into rural and urban regions during stratospheric-tropospheric folding events leading to increased background levels of O3 at the surface. 7Be can be used as a tracer of upper atmospheric air parcels and the O3 associated with them. Aerosol samples with a 2.5-microm cutoff were collected during 12-hr cycles (day/night) for a 30-day period at Deer Park, TX, near Houston, in August-September of 2000, and at Waddell, AZ, near Phoenix, in June-July of 2001. A comparison of 7Be levels with 12-hr O3 averages and maxima shows little correlation. Comparison of nighttime and daytime O3 levels indicate that during the day, when mixing is anticipated to be higher, the correlation of 7Be with O3 in Houston is approximately twice that observed at night. This is consistent with mixing and with the anticipated loss of O3 by reaction with nitric oxide (NO) and dry deposition. At best, 30% of the O3 variance can be explained by the correlation with 7Be for Houston, less than that for Phoenix where no significant correlation was seen. This result is consistent with the intercept values obtained for 7Be correlations with either O3 24-hr averages or O3 12-hr maxima and is also in the range of the low O3 levels (25 ppb) observed at Deer Park during a tropical storm event where the O3 is attributable primarily to background air masses. That is, maximum background O3 level contributions from stratospheric sources aloft are estimated to be in the range of 15-30 ppb in the Houston, TX, and Phoenix, AZ, area, and levels above these are because of local tropospheric photochemical production.
A 7BeCl2 solution containing 0.5 micrograms Be per mouse was injected subcutaneously, intraperitoneally, intramuscularly, intrathoracically, and intravenously, and distribution was observed for periods up to 1 wk. 7Be was excreted more rapidly following intravenous injection than by the other routes of injection. The amount of Be found in the liver or the spleen was substantial at 1 d after intraperitoneal injection. It increased more in the spleen at 7 d after either intraperitoneal or intrathoracic injection. On the other hand, the amounts of Be stayed almost constant in the kidneys, by the various routes of injection. When injected intrathoracically, the amounts of Be in the heart and the lung were greater than when administered by the other routes of injection. The amounts of Be in the femurs of mice administered by these routes of injection, except with intravenous injection, were greater than in the other organs. The percentage of 7Be in the mineralized bone was 90% of that of 7Be in the femurs when injected intraperitoneally or intrathoracically. However, the ratio of Be in the mineralized bone to that in the bone marrow was 3 to 2. Beryllium had thus a closer affinity for the femurs than for the other organs investigated, with the different modes of administration used. The amount of Be in the entire skeleton was estimated to be substantial. Within the limitations of 1 wk of exposure, the skeleton would appear to be a critical organ. This would suggest that osteosarcomas may occur following administration of Be to laboratory animals for a long-term period.
Measurements of dose rate and penetration in water have been made for neutron beams produced by 30--75 MeV protons on beryllium. The effects of Polythene filters added on the target side of the collimator have also been studied. A neutron beam comparable with a photon beam from a 4--8 MeV linear accelerator can be produced with p/Be neutrons plus 5 cm Polythene filtrations, with protons in the range 50--75 MeV. This is a more economical method than use of the d/Be reaction.
Lineal energy spectra of fast neutrons, produced by bombarding a thick beryllium target with 14 MeV deuterons, were measured with a proportional counter simulating a sensitive diamter of 2 micrometers. The mean neutron energy of the collimated beam was 6 MeV. The total lineal energy spectra, measured at different positions within a water phantom were separated into three different components: the gamma component, the recoil-proton component, and the component of heavier recoil ions. It is shown mathematically that the components of a linear energy spectrum thus derived allow the calculation of their fractional dose contributions. These dose fractions i.e. the gamma dose, the recoil-proton dose, and the dose fraction of heavier recoil ions, are shown as a function of the spatial position in the phantom.
Beryllium (Be) metal and several of its analogues have been shown to be carcinogenic in rats. In addition, workers employed at Be processing plants have been shown to have a slight excess of lung cancer. In this study, a single inhalation exposure to Be metal produced a 64% incidence of lung tumors in the F344/N rat. The most frequent tumor type observed was adenocarcinoma. These Be metal-induced lung carcinomas were examined for genetic alterations in the K-ras, p53, and c-raf-1 genes. DNA isolated from lung neoplasms was analyzed by PCR amplification and direct DNA sequence analysis, immunohistochemical analysis and Southern blot analysis. No K-ras codon 12, 13 or 61 mutations were detected in 24 lung tumors by direct sequencing. Using a more sensitive K-ras codon 12 mutation selection assay, K-ras codon 12 GGT-GTT transversions were detected in two of 12 adenocarcinomas. These results suggest that activation of the K-ras protooncogene is both a rare and late event, possibly stemming from genomic instability during the progression of some Be-induced rat adenocarcinomas of the lung. No mutant p53 nuclear immunoreactivity was observed in any Be-induced tumor. Because immunohistochemical analysis of the p53 protein only detects missense mutations, exons 5-8 of this gene were also analyzed by direct DNA sequencing. In order to perform the p53 sequence analysis, it was necessary to first characterize and sequence the p53 intron sequences flanking exons 5-8 and their splice sites. Details of this expanded intron DNA sequence information are given here. No mutations were detected within exons 5-8 of the p53 gene. No rearrangement of the c-raf-1 protooncogene was detected by Southern blot analysis. These results indicate that the mechanisms underlying the development of Be-induced lung cancer in rats do not involve gene dysfunctions commonly associated with human non-small-cell lung cancer.
Biological data is necessary for estimation of protection from neutrons, but there is a lack of data on biological effects of neutrons for radiation protection. Radiological study on fast neutrons has been done at the National Institute of Radiological Sciences. An intense neutron source has been produced by 25 MeV deuterons on a thick beryllium target. The neutron energy spectrum, which is essential for neutron energy deposition calculation, was measured from thermal to maximum energy range by using an organic liquid scintillator and multi-sphere moderated 3He proportional counters. The spectrum of the gamma rays accompanying the neutron beam was measured simultaneously with the neutron spectrum using the organic liquid scintillator. The transmission by the shield of the spurious neutrons originating from the target was measured to be less than 1% by using the organic liquid scintillator placed behind the collimator. The measured neutron energy spectrum is useful in dose calculations for radiobiology studies.
Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of insoluble beryllium (Be) dusts and characterized by the accumulation of CD4+ T cells and macrophages in the lower respiratory tract. In response to Be inhalation, noncaseating granuloma formation and, eventually, fibrosis. The immunopathogenic process is maintained by Be-specific lung CD4+ T-lymphocytes. Consistent with the disease immunopathology, these Be-specific T cells have a T-helper 1 phenotype producing interleukin-2 and interferon-gamma, the macrophage-activating cytokine driving the granulomatous reaction. Previous studies have demonstrated that the glutamic acid in position 69 of the human leukocyte antigen class II b chain is strongly associated with increased susceptibility to Be in exposed workers, suggesting that human leukocyte antigen gene markers may be used as epidemiological probes to identify population groups at higher risk.
Employing monoclonal antibodies that identify T-helper/inducer or T-suppressor/cytotoxic cells, we investigated the T-lymphocyte subsets in hypersensitivity granulomas developed after beryllium salt injection. T-helper/inducer cells were detected at all stages of the lesions, including early nonspecific inflammatory lesions seen at 3 weeks after injection. They formed clusters intermingled with the mononuclear phagocytes in granulomatous lesions, but were not found among the epithelioid cells. In contrast, T-suppressor/cytotoxic cells were slow to appear in the lesions and constituted very small numbers in mature epithelioid cell granulomas of 9-11 weeks. These findings suggest that T-helper/inducer cells may be responsible for the evolution of the perivascular infiltrate of mononuclear cells. Furthermore, appearance of T-helper/inducer cells without T-suppressor/cytotoxic cells may contribute to the development and organization of epithelioid cells and their maturity in the granulomatous lesions.
An improved vacuum evaporator is described for coating frozen-hydrated biological samples with beryllium for X-ray microanalysis. The evaporator permits repeated coatings without bringing the main chamber to atmospheric pressure and ambient temperature. The use of a glass sleeve in the evaporation chamber facilitates cleaning.
An increasing number of metals with the potential to cause allergic contact dermatitis have found their way into dental alloys for economic and practical reasons. 2 patients are reported who developed gingivitis adjacent to the Rexillium III alloy in their dental prostheses. Patch testing demonstrated positive reactions to beryllium sulfate, a component of the alloy. Components of dental alloys and the mechanism of the contact dermatitis are discussed.
Cells of spleen and lymph nodes were obtained from mice subcutaneously injected with BeCl2 once a week over a 6-week period. These cells were washed twice in cold phosphate-buffered saline and suspended at a final concentration of 2 x 10(7) cells in 1 ml of buffer. Subsequently, 0.5 ml of the suspension was injected into normal mice intravenously (passive transfer). The findings of the experiments using these mice are summarized as follows: (1) The footpad reaction test was performed 24 hrs after passive transfer. The mice receiving spleen cells or lymph node cells from mice sensitized with BeCl2 developed significant footpad swelling 24 and 48 hrs after challenge of BeCl2 as the antigen. (2) After passive transfer with sensitized spleen cells or lymph node cells the ears of the mice were painted with 7% BeCl2 in ethyl alcohol. Ears of these mice showed significant swelling as compared with those of controls. (3) The migration of peritoneal cells from mice receiving spleen or lymph node cells from sensitized mice was inhibited when tested in medium containing the antigen of BeCl2. From our present experiments, it was suggested that the beryllium sensitizing ability was related to active as well as passive cell-mediated immunity.
Protons of 35 and 65 MeV and deuterons of 35 MeV were used to bombard beryllium and lithium targets of various thicknesses. Four types of experiments were conducted in order to characterize the neutron fields. They were (1) central axis depth-dose measurements in a water phantom, (2) dose buildup at small depths in tissue-equivalent plastic, (3) microdosimetric measurements and LET distributions, and (4) neutron yields and energy spectra at an angle of 0 deg. The results generally show that (a) the central axis depth doses for the 35 and 65 MeV particles roughly approximate those of 60Co and 4-MeV bremsstrahlung photons, respectively, (B) the neutron dose buildups are more rapid than those of the above-mentioned photon sources, (C) the microdosimetric spectra show differences which are consistent with the measured neutron energy spectra, and (D) P-Li compared to p-Be neutron spectra have larger high-energy particle flux for similar target and beam configurations.
The production of neutrons by stopping 25-, 35-, 45-, and 55-MeV protons in beryllium has been studied. The yield of neutrons in the forward direction can be approximated by the relation: yield = -0.032X10(16).E+0.0027X10(16).E2 neutrons/C sr. The neutron spectra are characterized by a low-energy evaporation component which has small angular dependence and a high-energy component which is peaked in the forward direction. Neutrons produced in the forward direction have an average energy approximately 40% of the incident proton energy.