Radiosensitization in multifraction schedules. I. Evidence for an extremely low oxygen enhancement ratio

Stable monolayers of contact-inhibited C3H 10T1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.     

Construction of hybrid Tn501/Tn21 transposases in vivo: identification of a region of transposase conferring specificity of recognition of the 38-bp terminal inverted repeats.

In order to study the transposase enzymes of Class II prokaryotic transposable elements, we have constructed genes encoding hybrid transposase proteins. This was done by recombination in vivo between the tnpA genes of transposons Tn501 and Tn21. These hybrid genes can complement in trans a transposition-defective mutant of Tn501. The structures of the products of this complementation indicate whether the specificity of the hybrid transposase in recognising the 38 bp terminal inverted repeats is that of Tn501 or that of Tn21. The determinant of this specificity is in the N-terminal region of the transposase protein, between amino acids 28 and 216. The predicted amino acid sequences so far determined of transposases from the Class II family reveal an area of homology in this region.     

Binding of a nuclear factor to a consensus sequence in the 5' flanking region of zein genes from maize

The genomic organization of the zein structural genes and of regulatory loci influencing their expression suggests that control of zein gene expression will involve interactions between cis elements in the flanking DNA sequences and products from trans-acting genes. The interaction between fragments from the 5' flanking region of a zein gene and specific, double-stranded oligonucleotides with crude nuclear extracts from maize endosperm have been studied by nitrocellulose filter binding, gel retention and DNase I footprinting assays. Specific binding of a nuclear factor was observed and the exact position of the protein binding site was determined. The 22-nt binding site included 14 bp of a 15-bp sequence conserved in all zein genes.     

Proteoglycan metabolism associated with mouse metanephric development: morphologic and biochemical effects of beta-D-xyloside

Morphology and de novo incorporation of [35S]sulfate into proteoglycans were studied in fetal mouse kidneys at the onset of organogenesis. Branching morphogenesis and nephron development in organ culture and in vivo were associated with de novo synthesis of chondroitin-SO4 and heparan-SO4 proteoglycans. The role of proteoglycan metabolism in metanephrogenesis was then studied by analysis of the effects of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside) on renal development and proteoglycan metabolism. Incubation of fetal kidneys in beta-D-xyloside at concentrations of 1.0 and 0.5 mM, but not at 0.1 mM, caused inhibition of ureteric branching and markedly diminished synthesis of a large Mr 2.0 X 10(6) Da chondroitin-SO4 proteoglycan. Incorporation of [35S]sulfate was stimulated at all beta-D-xyloside concentrations, reflecting synthesis of xyloside initiated dermatan-35SO4 chains. In contrast to dramatic effects on chondroitin-SO4 synthesis and ureteric branching, beta-D-xyloside had no effect on heparan-SO4 synthesis or on development of the glomerulus and glomerular basement membrane. We thus characterize the proteoglycans synthesized early in the course of renal organogenesis and describe observations which suggest an association between metabolism of chondroitin-SO4 proteoglycan and development of the ureter.     

Locations of the hydrogenases of Methanobacterium formicicum after subcellular fractionation of cell extract.

The F420 hydrogenase of Methanobacterium formicicum was associated with membranes isolated by sucrose density gradient ultracentrifugation of cell extract. The methyl viologen hydrogenase was present in the soluble fractions. Column chromatography with phenyl-Sepharose CL-4B revealed that the F420 hydrogenase was strongly hydrophobic, suggesting that it associates with isolated membranes through hydrophobic interactions.     

Cholera toxin and pertussis toxin regulate the Fc receptor-mediated phagocytic response of human neutrophils in a manner analogous to regulation by monoclonal antibody 1C2

Data presented in this paper indicate that polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis can be markedly augmented and that this augmentation is under regulatory control. Stimulation of PMN with either a low m.w., heat-labile cytokine(s) (the culture supernatant effluent from a YM-10 Centricon unit, YM-10E), phorbol esters (phorbol dibutyrate), or the polyene antibiotic, amphotericin B, enhances Fc-mediated ingestion in a dose-dependent manner. YM-10 effluent- and amphotericin B-stimulated ingestion is completely abrogated by treating the PMN with either pertussis toxin (PT), cholera toxin (CT), or a monoclonal antibody (mAb), 1C2. However, neither toxin nor mAb 1C2 affects nonstimulated ingestion or phagocytosis stimulated by phorbol esters or synthetic diacylglycerol. Increasing intracellular cyclic adenosine monophosphate levels by stimulation with prostaglandin E1 and the phosphodiesterase inhibitor, isobutylmethylxanthine, does not mimic the effect of either toxin or mAb 1C2. In addition, toxin-mediated inhibition is not due to loss of either the Fc receptor recognized by mAb 3G8 or the antigen recognized by mAb 1C2. These data indicate that both CT and PT regulate the phagocytic response of PMN, in a manner like mAb 1C2, probably by affecting a guanosine 5'-triphosphate-binding protein distinct from those that regulate adenylate cyclase. Since phorbol ester-stimulated ingestion is not inhibited by either PT, CT, or mAb 1C2 and phorbol esters activate protein kinase C directly, phagocytosis amplification regulated by PT, CT, and mAb 1C2 may involve protein kinase C activation.     

Molecular dynamics simulations of "loop closing" in the enzyme triose phosphate isomerase

We present molecular dynamics simulations on the active site region of dimeric triose phosphate isomerase (TIM) using the co-ordinates of native chicken muscle TIM as a starting point and performing simulations with no substrate, with dihydroxyacetone phosphate (DHAP), the natural substrate, and with dihydroxyacetone sulfate (DHAS), a substrate analog. Whereas most of the protein moves less than 1 A during the simulation, some residues in the active site loop move more than 8 A during the 10.5 picoseconds of dynamics for each of the simulations. Most interestingly, the nature of the loop motion depends on the substrate, with the largest motion found in the presence of DHAP, and only in the presence of DHAP does the loop move to "close off" the active site pocket. The final structure found for the DHAP-chicken TIM complex is qualitatively similar to that described by Alber et al. for DHAP-yeast TIM. Simulations on the monomeric protein gives insight into why the molecule is active only as a dimer.     

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)