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PubMed1988

Ligand-induced structural constraints in human dihydrofolate reductase revealed by peptide-specific antibodies.

Peptides from human dihydrofolate reductase (DHFR) generated by cyanogen bromide cleavage and corresponding to residues 15-52, 53-111, 112-125, and 140-186 (carboxyl terminus) were purified and used to immunize rats. Titration of the immune sera against denatured human DHFR by solid-phase immunoassay showed that peptides 15-52 and 140-186 were relatively highly immunogenic, unlike the native enzyme which is most immunogenic in the sequence 53-111. The antisera were specific for the corresponding peptides used for immunization. Antibodies to peptides 15-52, 53-111, and 140-186 cross-reacted with native human DHFR in solution in competition assays. However, the binding of nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and the inhibitors folate and methotrexate, both in binary and in ternary complexes with the enzyme, caused a striking reduction in binding of antibody. Using a sensitive radioactive assay, it was found that antisera to peptides 15-52 and 140-186, both of which exhibited a high antibody titer, caused significant inhibition of DHFR. Because peptide 140-186 does not include any active-site residues, it is concluded that at least in this case all the antibodies bound to regions outside the active site. Since comparison of the X-ray structures of the chicken liver DHFR holoenzyme with the apoenzyme reveals no changes in secondary structural elements (alpha-helices and beta-sheets), the reduction in antibody binding to DHFR-ligand complexes must not involve epitopes within these structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗

PubMed1986

Comparative studies of selenium-75 (selenite and selenomethionine) absorption from various milk diets in suckling rats.

Selenium (Se) absorption was studied in human milk, bovine milk and infant formula (Similac) using suckling rats as a model. The effect of age on Se absorption from the three milk diets extrinsically labeled with 75Se, either as selenite or selenomethionine, was also investigated. Milk diets were fed by gastric intubation and the radioactivity in the carcass, gastrointestinal tract and the liver were measured 3 h after feeding. There was no difference in [75Se]selenite absorption from the three milk diets between 8-20 d of age. However, significantly higher quantity of 75Se was absorbed from all three milk diets by 20-d-old rats than by the younger rats (46 vs. 32%). This increase in [75Se]selenite absorption with advancing age is opposite to what has been found for most other trace elements. When rats were fed milk diets labeled with [75Se]selenomethionine, the absorption of 75Se was approximately twofold higher in all age groups compared with 75Se absorption from selenite. No difference in [75Se]selenomethionine absorption existed among the three milk diets in 8- or 10-d-old suckling rats. However, at 15 d of age [75Se]selenomethionine absorption from human milk was higher (82%) than from either bovine milk (72%) or infant formula (72%). Between 8 and 20 d of age, absorption of [75Se]selenomethionine from the three milk diets decreased with advancing age. Adding sodium selenate to increase the total nonradioactive Se of human milk, bovine milk (endogenous plus the added selenium) did not affect the absorption of either [75Se]selenomethionine or [75Se]selenite.

Aging↗

PubMed1995

A comparison of techniques used to estimate the amount of resuspended soil on plant surfaces.

The objectives of this study were to compare four common techniques used to estimate soil mass loadings on plant surfaces and to assess the need to account for particle-size distributions of both the soil tracer and contaminant of concern within the soil. Soil loadings (g soil kg-1 dried plant) from split samples collected in a pasture near Chernobyl were estimated using soil tracers of plutonium analyzed via alpha spectroscopy (mean +/- standard error; 1.0 +/- 0.2), titanium analyzed with an inductive coupled plasma spectrometer; (3.6 +/- 0.6), and neutron activation analysis for scandium (8.1 +/- 1.6), as well as simply washing the soil off the vegetation (34.1 +/- 5.6) Differences were significant at p < 0.001. We also found that soil loading estimates from any one technique varied by a factor of 10 depending on the soil particle size used in the calculations. This was because soil loadings decreased when smaller-sized soil fractions dominated the resuspension process. However, the percent of the plant's total contamination attributable to soil loading increased with smaller soil particles. Smaller soil particles apparently contribute less to the mass of soil loading (g soil kg-1 dry plant), but more to the total plant contamination (Bq) because of the higher concentration of contaminant found in the smaller-sized soil fractions. Differences in mass loading estimates due to the technique chosen (a factor of 10), or due to differences in elemental concentration as a result of the soil particle size used in the calculation (also a factor of 10), were greater than the natural variability observed in the field (2.5).

Alpha Particles↗

PubMed2003

Simulating needle insertion and radioactive seed implantation for prostate brachytherapy.

We are developing a simulation of needle insertion and radioactive seed implantation to facilitate surgeon training and planning for brachytherapy for treating prostate cancer. Inserting a needle into soft tissues causes the tissues to displace and deform: ignoring these effects during seed implantation leads to imprecise seed placements. Surgeons should learn to compensate for these effects so seeds are implanted close to their pre-planned locations. We describe a new 2-D dynamic FEM model based on a 7-phase insertion sequence where the mesh is updated to maintain element boundaries along the needle shaft. The locations of seed implants are predicted as the tissue deforms. The simulation, which achieves 24 frames per second using a 1250 triangular element mesh on a 750Mhz Pentium III PC, is available for surgeon testing by contacting ron@ieor.berkeley.edu.

Brachytherapy↗

PubMed1987

[Beta-alanylation, a means for neutralization of histamine in the central nervous system of Carcinus maenas].

Histamine neutralization in Carcinus maenas is not ensured by oxidation, methylation, or acetylation. After injecting labelled histamine, radioactive carcinine (beta-alanylhistamine), biosynthesized in the central nervous system, rapidly accumulates in the heart. This synthesis is intense and proportional to the amount of histamine injected; on the contrary, it is very low after injecting labelled beta-alanine, whatever the amount injected. Ten days after injecting [14C]histamine, the amounts of radioactive carcinine stocked in the heart remain high. When incubated in the presence of labelled carcinine, various Carcinus tissues are unable to metabolize it. Thus it appears that carcinine would be the catabolite of histamine in Carcinus maenas and that beta-alanylation would be a novel pathway for histamine neutralization. Since carcinine synthetase activity is very high in the central nervous system, this enzyme might neutralize not only neuronal histamine, but also possibly exogenous histamine; thus it would constitute an element of the blood-brain barrier.

Alanine↗

PubMed2002

Mechanisms of desorption of 134Cs and 85Sr aerosols deposited on urban surfaces.

The radioactive isotopes of cesium and strontium may be deposited on urban surfaces in the case of an accidental atmospheric discharge from a nuclear facility and thus imply a health hazard. In order to handle the decontamination of these surfaces, we have carried out experiments under controlled conditions on tiles and concrete and we have studied.the physical and chemical mechanisms at the solid-liquid interface. The deposition of radionuclides was carried out in the form of aerosols indicating an accidental source term. Their desorption by rainwater is low in all cases, of the order of 5-6% for cesium for any material and 29 and 12% for strontium on tile and concrete, respectively. The low desorption values of cesium may be explained by the strong bonding that occurs with the silicates constituting the tile due to virtually irreversible processes of exchange of ions and by the formation of insoluble complexes with the C--S-H gel of concrete. The strontium-tile bonds are weaker, while strontium precipitates with the carbonates of concrete in the form of SrCO3. In view of these characteristics, washing solutions with high concentrations of chloride and oxalate of ammonium chosen for their ion-exchanging and sequestering properties were tested on these surfaces. The desorption of cesium improved strongly since it reached 70% on tile and 90% on concrete after 24h of contact, which is consistent with our knowledge of the; bonds between this element and the surfaces. Strontium, given the greater complexity of physical and chemical forms that it may take is less well desorbed. The ammonium chloride improves the desorption (50% and 40%, for tile and concrete, respectively) but the oxalate, while it does not affect desorption on the tiles, decreases that on the concrete since by strongly etching the concrete, it causes the release of carbonate ions that precipitate with strontium.

Adsorption↗

PubMed1995

Differentially expressed genes after peripheral nerve injury.

In an attempt to identify genes associated with Wallerian degeneration and peripheral nerve regeneration we have performed differential hybridization screening of a cDNA library from crushed rat sciatic nerve (7 days postlesion) using radioactively labeled cDNA prepared from poly(A)+ RNA of normal vs. crushed nerve. Screening of 5,000 randomly selected colonies yielded 24 distinct clones that were regulated following nerve injury. Fifteen of the differentially expressed sequences could be classified as induced, whereas 9 sequences appeared to be repressed at 1 week postcrush. Sequencing and computer-assisted sequence comparison revealed 3 classes of regulated cDNA clones representing 1) novel gene sequences (8 clones) including 3 transcripts containing a repetitive "brain identifier" (ID) element; 2) identified genes (7 clones) with previously undetected expression in the peripheral nervous system (PNS), such as apolipoprotein D, peripheral myelin protein 22kD (PMP22), SPARC (secreted protein, acidic and rich in cysteine), sulfated glycoprotein SGP-1, apoferritin, decorin, and X16/SRp20; and 3) identified genes (9 clones) with known expression in the PNS including, e.g., the myelin protein P0, gamma-actin, vimentin, alpha-tubulin, chargerin II, and cytochrome c-oxidase subunit I. Northern blot and polymerase chain reaction analyses with RNA from crushed and transected nerve demonstrated that sequences with related function, like the group of myelin genes, cytoskeleton genes, genes involved in RNA processing and translation, in lipid transport or energy metabolism showed closely related temporal patterns of expression during nerve degeneration and regeneration. Finally, we compared the differentially expressed genes identified at 7 days after crush injury (this investigation) with the regulated sequences isolated previously by De Leon et al. (J Neurosci Res 29:437-488, 1991) from a 3 day postcrush sciatic nerve cDNA library.

Animals↗

PubMedDate not supplied

Density distribution of guinea pig myenteric plexus nerve endings containing immunoreactive substance P.

The present study was performed to investigate how myenteric plexus nerve endings containing substance P are distributed in sucrose density gradients in relation to nerve endings capable of taking up 3H-acetylcholine or 14C-noradrenaline. The peak of substance P-immunoreactivity (ISP) was found at a density of 1.157 +/- 0.001 g X ml-1, that of 3H-radioactivity at 1.160 +/- 0.002 and that of 14C-radioactivity at 1.162 +/- 0.002 g X ml-1 (mean +/- SEM, N = 6); there was considerable overlap. In a second set of experiments, the resuspended P2-pellet was layered upon a discontinuous density gradient consisting of 0.6, 1.0, 1.2 and 1.4 M sucrose. Nine fractions were recovered. There was a 2.5-3.4-fold increase in the relative specific activity of ISP in the 1.2 M fraction (density = 1.154 g X ml-1) and the adjoining interfaces. Conventional electron microscopy showed that synaptosomal elements were present in the transmitter-enriched fractions. It is concluded that the substance P-containing nerve endings of the guinea pig myenteric plexus co-distribute (and may be co-purified with) nerve endings utilizing noradrenaline or acetylcholine on sucrose density gradients.

Acetylcholine↗

PubMed2000

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells.

This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.

Anti-Bacterial Agents↗

PubMed1985

Deletion of a yeast small nuclear RNA gene impairs growth.

We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.

Base Sequence↗

PubMed1997

Comparison of the breakpoint regions of ELE1 and RET genes involved in the generation of RET/PTC3 oncogene in sporadic and in radiation-associated papillary thyroid carcinomas.

The RET/PTC3 oncogene is an activated form of the RET protooncogene, which is frequently rearranged in papillary thyroid carcinoma. RET/PTC3 results from a structural rearrangement between the ELE1 and the RET genes, and it has been observed in both sporadic and radiation-associated post-Chernobyl tumors. To understand the molecular basis that predisposes RET and ELE1 genes to be recurrent targets of "illegitimate" recombination, we examined the genomic regions containing the ELE1/RET breakpoints of six sporadic and three post-Chernobyl tumors in two papillary carcinomas of different origins. Our data indicated, in both genes, a clustering of the breakpoints in regions designated ELE1-bcr (1.8 kb) and RET-bcr (1.9 kb). Notably, in all sporadic tumors and in one post-Chernobyl tumor the ELE1/RET recombination corresponded with short sequences of homology (3-7 nt) between the two rearranging genes. In addition, we observed an interesting distribution of the post-Chernobyl breakpoints in ELE1-bcr located within an Alu element, or in between two close Alu elements, and always in A+T-rich regions.

Base Sequence↗

PubMed1992

Molecular analysis of APC mutations in familial adenomatous polyposis and sporadic colon carcinomas.

Mutations in the APC gene give rise to familial adenomatous polyposis (FAP) and also occur in many, perhaps most, sporadic colon cancers. By screening with single-strand conformation polymorphism analysis we identified several mutations in a small region of the APC gene in both FAP and sporadic cancers. These mutations were either point mutations or small deletions or insertions causing frameshifts, and all generated stop codons. One 5 base-pair deletion was found in a sporadic colon tumour, a colorectal cancer cell line derived from a sporadic colon tumour, and in four unrelated FAP patients. This mutation produces distinctive heteroduplex bands, which can be detected with a simple non-radioactive assay. Our findings suggest that highly localised short sequences, essentially runs that code for adenine and thymine, may account for up to 20% of all observed APC mutations.

Adenomatous Polyposis Coli↗

PubMed1985

The terminations of corticospinal tract axons in the macaque monkey.

This study examined the corticospinal tract in monkey by utilizing the anterograde transport of wheat germ lectin conjugated to horseradish peroxidase (WGA HRP) at the light microscopic level and the axonal transport of 3H-proteins with both light and electron microscopic autoradiographic techniques. The animals survived 3-9 days after the injections of 3H-leucine or 3H-leucine/WGA HRP into either motor or sensory cortices. With the laminar schema of Rexed as a guide to the layers of the spinal gray matter, qualitative and quantitative analyses of labeled projections of the corticospinal tract (CST) were made. With the light microscope, axons from the sensory cortex labeled with WGA-HRP could be observed in the contralateral spinal gray from lamina I to the border of laminae VI/VII, the heaviest distribution being located in medial III-VI. There was a small ipsilateral projection to V and VI. With 3H label, laminae I and II revealed few overlying silver grains; many grains overlay laminae III-VI. Projections from the motor cortex labeled with either WGA-HRP or 3H extended from the contralateral laminae III/IV border into the motor nucleus (lamina IX) and were seen to be somewhat more dense in the lateral areas of the spinal gray. The motor cortex projected heavily to ipsilateral VIII, and in sparse amounts to ipsilateral V and VI. Electron microscopy of radioactive axons from the sensory cortex to dorsal horn revealed many radioactive myelinated fibers and some labeled non-myelinated axons. Labeled terminals contacted medium to small dendrites; there were a few labeled C-type profiles in glomeruli and occasional axo-axonal or dendro-axonal contacts, the labeled cortical axons being the postsynaptic structure. In ventral horn following motor cortex injections, the labeled axons were all myelinated. The synaptic contacts were found on small, medium, and large proximal dendrites as well as on cell bodies. Labeled terminals which formed the central element in glomeruli were also seen in this region. Most of the labeled corticospinal terminals in dorsal and ventral horn contained rounded vesicles, but a significant number revealed pleomorphic vesicles. The relationship of these morphological findings to physiological studies of the CST is presented.

Animals↗

PubMed1999

Cleavage of DNA by human NM23-H2/nucleoside diphosphate kinase involves formation of a covalent protein-DNA complex.

The NM23 gene family in humans is implicated in differentiation and cancer, but the biochemical mechanisms are unknown. Most NM23 proteins have phosphotransferase (nucleoside diphosphate kinase) activity, and the second human isoform, NM23-H2, also binds to a nuclease-hypersensitive c-MYC promoter element through which it activates c-MYC transcription. It is shown here that this DNA binding can result in double-stranded breaks. The DNA breaks occur within repeated sequence elements in the linear nuclease-hypersensitive duplex and leave staggered ends with 5-nucleotide-long 3'-extensions. The enzyme also cleaves supercoiled plasmid DNA to yield nicked circular and unit length linear products. The cleavage reaction requires only NM23-H2, DNA, Mg(2+), and buffer, occurs in the absence of denaturing conditions, and can be reversed by EDTA. The cleaved DNA strands have free 3'-OH groups, and protein is attached to the 5'-phosphoryl ends. Transfer of (32)P radioactivity from DNA to NM23-H2 has been observed, and a covalent polypeptide-DNA complex has been isolated and identified by Western blotting as NM23-H2. Since covalent protein-DNA complexes are known to serve the role of breaking and rejoining DNA strands, the present findings suggest that NM23-H2 is involved in DNA structural transactions necessary for the activity of the c-MYC promoter.

Antigens, Neoplasm↗

PubMed1985

Terminal repetitive sequences in herpesvirus saimiri virion DNA.

The H-DNA repeat unit of Herpesvirus saimiri strain 11 was cloned in plasmid vector pAGO, and the nucleotide sequence was determined by the dideoxy chain termination method. One unit of repetitive DNA has 1,444 base pairs with 70.8% G+C content. The structural features of repeat DNA sequences at the termini of intact virion M-DNA (160 kilobases) and orientation of reiterated DNA were analyzed by radioactive end labeling of M-DNA, followed by cleavage of the end fragments with restriction endonucleases. The termini appeared to be blunt ended with a 5'-phosphate group, probably generated during encapsidation by cleavage in the immediate vicinity of the single ApaI recognition site in the H-DNA repeat unit. The sequence did not reveal sizeable open reading frames, the longest hypothetical peptide from H-DNA being 85 amino acids. There was no evidence for an mRNA promoter or terminator element, and H-DNA-specific transcription could not be found in productively infected cells.

Animals↗

PubMed1999

Nonsaturable entry of neuropeptide Y into brain.

Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 x 10(-4) ml. g. -1. min-1. However, this rate of entry was not significantly changed by injection of 10 microgram/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB.

Animals↗

PubMed1997

Analysis of the in vitro secondary structure of cucumber mosaic virus satellite RNA.

The solution conformation of two variants of cucumber mosaic virus satellite RNA (CMV satRNA) was analyzed using several enzymatic and chemical probes. Ribonuclease T1 and nuclease S1 were used to map unpaired nucleotides, and nuclease V1 was used to detect double-stranded, or stacked, bases. Chemical probing with dimethylsulphate and diethylpyrocarbonate also identified unpaired and unstacked nucleotides, respectively. Modified or cleaved positions were identified by direct gel electrophoresis of radioactively labeled RNA, or by analysis of DNA sequence patterns generated by primer-extension with reverse transcriptase. Additional information was obtained by a gel-fractionation method under nondenaturing conditions for the identification of base paired fragments. On these data, a model for the in vitro secondary structure of CMV satRNA is proposed. Results support the existence of a complex structure with 51% of nucleotides involved in base pairs (40 G:C, 28 G:U, and 18 A:U pairs). Several structural elements, numbered I-VI, were defined, and interactions between separate domains are suggested. Comparisons of experimental data and a formerly reported secondary structure model for CMV satRNA support the validity of the structure we propose.

Base Composition↗

PubMed1989

[The transfer of 137Cs and 90Sr into flour, bran and straw from wheat, rye, barley and oats in the years 1982, 1986 (reactor accident in Chernobyl) and 1987 in field studies].

The specific activity of 137Cs and 90Sr from the global fallout, as well as from the fallout after the reactor accident at Chernobyl, was determined in flour, bran and straw from wheat, rye, barley and oats as well in the corresponding soils (Cambisol). The results show that the activity of 137Cs, but not of 90Sr, in the plant material was considerably higher in 1986, and still to some extent in 1987, compared to 1982. For 137Cs an 90Sr, as well as for most cereal samples, the activity in the bran and straw was significantly higher than in the flour. Determination of stable potassium and calcium in all samples revealed that this enrichment is, to a large extent, the result of a comparable enrichment of these elements in brans and straw. The plant/soil concentration ratios, averaged over all cereals, were for 137Cs (1982 and 1987): flour 0.026 +/- 0.018; bran 0.079 +/- 0.042; straw 0.055 +/- 0.027. For 90Sr (1982, 1986, and 1987): flour 0.19 +/- 0.095; bran 0.70 +/- 0.23; straw 2.35 +/- 0.82.

Accidents↗