The sensitivities of SV40-transformed human fibroblasts to monofunctional and DNA-crosslinking alkylating agents.

4 repair-deficient (Mer-) and 2 repair-proficient (Mer+) lines of SV40-transformed human fibroblasts were assayed for colony-forming ability after treatment with MNNG, methyl methanesulfonate (MMS), 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), and 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU). The sensitivities to MMS, BCNU and HECNU of these SV40-transformed lines were similar to those of comparably treated human tumor cells observed previously. However, unlike human tumor lines, whose post-MNNG survival is strongly dependent upon Mer phenotype, SV40-transformed lines showed a lack of dependence of post-MNNG colony-forming ability on Mer phenotype. No differences in glutathione levels that might explain these differences were detected. The amounts of SV40-specific DNA and RNA among the lines were found to vary widely, but no correlation with Mer phenotype was found.     

Limitations of Photosynthesis in Pinus taeda L. (Loblolly Pine) at Low Soil Temperatures

The relative importance of stomatal and nonstomatal limitations to net photosynthesis (A) and possible signals responsible for stomatal limitations were investigated in unhardened Pinus taeda seedlings at low soil temperatures. After 2 days at soil temperatures between 13 and 7°C, A was reduced by 20 to 50%, respectively. The reduction in A at these moderate root-chilling conditions appeared to be the result of stomatal limitations, based on the decrease in intercellular CO₂ concentrations (c_i). This conclusion was supported by A versus c_i analysis and measurements of O₂ evolution at saturating CO₂, which suggested increases in stomatal but not biochemical limitations at these soil temperatures. Nonuniform stomatal apertures, which were demonstrated with abscisic acid, were not apparent 2 days after root chilling, and results of our A versus c_i analysis appear valid. Bulk shoot water potential (ψ) declined as soil temperature dropped below 16°C. When half the root system of seedlings was chilled, shoot ψ and gas-exchange rates did not decline. Thus, nonhydraulic root-shoot signals were not implicated in stomatal limitations. The initial decrease in leaf conductance to water vapor after root chilling appeared to precede any detectable decrease in bulk fascicle ψ, but may be in response to a decrease in turgor of epidermal cells. These reductions in leaf conductance to water vapor, which occurred within 30 minutes of root chilling, could be delayed and temporarily reversed by reducing the leaf-to-air vapor-pressure deficit, suggesting that hydraulic signals may be involved in initiating stomatal closure. By independently manipulating the leaf-to-air vapor-pressure deficit of individual fascicles, we could induce uptake of water vapor through stomata, suggesting that nonsaturated conditions occur in the intercellular airspaces. There was an anomaly in our results on seedlings maintained for 2 days at soil temperatures below 7°C. Lower A appeared primarily the result of nonstomatal limitations, based on large increases in calculated c_i and A versus c_i analysis. In contrast, measurements of O₂ evolution at saturating CO₂ concentrations implied nonstomatal limitations per se did not increase at these temperatures. One explanation for this paradox is that calculations of c_i are unreliable at very low gas-exchange rates because of inadequate measurement resolution, and limitations of A are predominantly stomatal. An alternative interpretation is that increases in c_i are real and the results from O₂-evolution measurements are in error. The high CO₂ concentration used in O₂-evolution measurements (15%) may have overcome nonstomatal limitations by enzymes that were down-regulated by a feedback mechanism. In this scenario, carbohydrate feedback limitations may be responsible for nonstomatal reductions in A after 2 days at soil temperatures below 7°C.     

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.     

X-ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, have been determined by X-ray crystallography to a resolution of 1.8 A. Crystals of this rubredoxin grow in space group P2(1)2(1)2(1) with room temperature cell dimensions a = 34.6 A, b = 35.5 A, and c = 44.4 A. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X-PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 A and 2.06 degrees, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 A and in bond angles of 1.95 degrees. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen-bonding network in the beta-sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino-terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.     

Magnetization and electron paramagnetic resonance studies of reduced uteroferrin and its "EPR-silent" phosphate complex

The exchange coupling of reduced uteroferrin has been measured (19.8(5) cm-1 S1.S2) using recently developed techniques for studying metalloprotein magnetization. A spin Hamiltonian describing the coupled binuclear Fe(II).Fe(III) center has been used to fit the low and high field magnetization data, the EPR g values, and the highly anisotropic effective hyperfine tensor of the ferric site. The exchange coupling of the phosphate complex of reduced uteroferrin has also been measured (6.0(5) cm-1 S1.S2) using the same techniques. The smaller exchange coupling of the phosphate complex is comparable with the zero field splittings of the iron sites. This results in increased sensitivity of the system g values (found by calculation from the spin Hamiltonian) to variations of the zero field splitting parameters arising from heterogeneities in the protein microenvironment. Consequently, there is a very significant (9-fold) increase in the "effective g strain" of the system compared to the situation in the absence of phosphate. This, together with the larger g anisotropy (g = (1.06, 1.51, 2.27)), gives rise to an EPR signal for the phosphate complex of reduced uteroferrin which is extremely broad and difficult to detect but which has now been identified for the first time.     

A theory of the symmetries of filamentous bacteriophages.

A mathematical model is presented which explains the symmetries observed for the protein coats of filamentous bacterial viruses. Three viruses (Ff, IKe, and If1) all have five-start helices with rotation angles of 36 degrees and axial translations of 16 A (Type I symmetry), and three other viruses (Pf1, Xf, and Pf3) all have one-start helices with rotation angles of approximately equal to 67 degrees and translations of approximately 3 A (Type II symmetry). The coat protein subunits in each group diverge from each other in amino acid sequence, and Type II viruses differ dramatically in DNA structure. Regardless of the differences, both Type I and Type II symmetry can be understood as direct, natural consequences of the close-packing of alpha-helical protein subunits. In our treatment, an alpha-helical subunit is modeled as consisting of two interconnected, flexible tubular segments that follow helical paths around the DNA, one in an inner layer and the other in an outer layer. The mathematical model is a set of algebraic equations describing the disposition of the flexible segments. Solutions are described by newly introduced symmetry indices and other parameters. An exhaustive survey over the range of indices has produced a library of all structures that are geometrically feasible within our modeling scheme. Solutions which correspond in their rotation angles to Type I and Type II viruses occur over large ranges of the parameter space. A few solutions with other symmetries are also allowed, and viruses with these symmetries may exist in nature. One solution to the set of equations, obtained without any recourse to the x-ray data, yields a calculated x-ray diffraction pattern for Pf1 which compares reasonably with experimental patterns. The close-packing geometry we have used helps explain the near constant linear mass density of known filamentous phages. Helicoid, rigid cylinder, and maximum entropy structure models proposed by others for Pf1 are reconciled with the flexible tube models and with one another.     

Guinea pig and gerbil erythrocytes rosette with different cells in the blood, bone marrow, and thymus of the cat

Cat thymocytes, bone marrow cells, and peripheral blood leukocytes (PBL) formed rosettes with guinea pig (GP) and gerbil (G) erythrocytes (E). In PBL from adult cats the frequency of rosettes was 27% with GPE and GE, while an average of 33% bone marrow cells formed rosettes with GPE and only 4% with GE. Thymocytes from kittens showed a high percentage of rosettes with both GPE and GE (35 to 81%), with the frequency of each type varying with the thymus tested. Fluorescein isothiocyanate labeling of one of the erythrocyte species revealed these cells to be rosetting with different nucleated cells; i.e., a low percentage (3-5%) of the rosettes formed with PBL and bone marrow had both labeled and unlabeled erythrocytes. In contrast, "mixed" rosettes were observed with a significant number of thymocytes, averaging 33% of thymocytes from six animals. A further distinction between the GE- and GPE-rosetting cells was revealed by a monoclonal antibody which blocked GE rosette formation without interfering with the binding of GPE to PBL and thymocytes. PBL could be depleted of either GPE- or GE-rosetting cells, with retention of IgG+ cells and cells capable of rosetting with the second erythrocyte species in the nonrosetting fractions. Stimulation of the latter nonrosetting fractions with pokeweed mitogen for induction of Ig synthesis revealed a T-lymphocyte specificity of the GE- and GPE-rosetting cells. PBL depleted of GE-rosetting cells yielded an increased Ig production, two- to threefold above the control; in contrast, depletion of GPE-rosetting cells from PBL resulted in a failure of the remaining cells to respond. These results suggest that T-suppressor cells of the cat are contained in the GE-rosetting fraction and T-helper cells are rosetted with GPE.     

Subpopulations of mature murine thymocytes: properties of CD4-CD8+ and CD4+CD8- thymocytes lacking the heat-stable antigen

The heat-stable antigen (HSA), recognized by the monoclonal antibodies M1/69, B2A2, and J11d, is low or absent on the surface of most murine peripheral T cells but present on all but 3% of thymocytes. The CD4-CD8+ and CD4+CD8- or "single positive" thymic populations may be divided into further subgroups based on surface HSA expression. One group, CD4-CD8+ and expressing very high levels of HSA (HSA++), is an immature, T cell antigen receptor (TcR) negative, outer cortical blast cell. However, a further subdivision of CD4-CD8+ and CD4+CD8- single positives may be made, into those negative to low for HSA (HSA-) and those expressing moderate amounts of HSA (HSA+). The proportion of HSA- single positives is low in the thymus of young mice, whereas the proportion of HSA+ single positives is similar to that of the adult. Both the HSA- and the HSA+ subsets of single positive thymocytes from adult mice are CD3+ and express the normal peripheral T cell incidence of V beta 8 determinants on the TcR. On stimulation with concanavalin A in limit-dilution culture both HSA- and HSA+ subsets of single positive thymocytes give a high frequency of proliferating clones, and the clones from both HSA- and HSA+ subsets of CD4-CD8+ thymocytes are cytotoxic. Thus both HSA- and HSA+ single positive thymocytes are functionally mature. The HSA- subsets of single positive thymocytes differ from the HSA+ subsets in being slightly larger in size, in expressing higher levels of MEL-14, in binding more peanut agglutinin, and in including a proportion of cells expressing high levels of the Pgp-1 glycoprotein. It is suggested that HSA- CD4-CD8+ and HSA- CD4+CD8- thymocytes are more mature than their HSA+ counterparts, and might represent a previously activated or "memory" thymic subpopulation.     

Influence of dietary-induced weight changes on serum luteinizing hormone, estrogen and progesterone in the bovine female

The working hypothesis in the present study was that changes in concentrations and secretory patterns of luteinizing hormone (LH), 17 beta estradiol (E2), and progesterone in sexually mature beef heifers fed diets deficient in energy are related to changes in body weight of the animals. Another important component of the study was to determine if concentrations and secretion patterns of the reproductive hormones changed over time as feeding of the experimental diets continued. Twelve Red Angus X Hereford heifers (20 mo of age; 355 +/- 7 kg) were assigned randomly to receive a low- (L, n = 7) or high- (H, n = 5) energy diet for 100 days (Day 0 = day of initiation of dietary treatment). All heifers were exhibiting estrous cycles at regular intervals when the experiment was initiated and continued to exhibit estrous cycles at regular intervals throughout the study. Stage of the estrous cycle was synchronized in all 12 heifers by administration of prostaglandin F2 alpha (PGF2 alpha) on two occasions (Days 45 and 75) during the experiment. Serial blood samples (taken at 12-min intervals for 4 h) were collected at 0, 12, 24, 36, 48, and 60 h after the PGF2 alpha injections (Days 45-47 and 75-77) to determine patterns of LH secretion during the follicular phase of the estrous cycle. In addition, serial blood samples (taken at 20-min intervals for 18 h) to monitor LH secretion during the luteal phase of the estrous cycle, in which the stage of the cycle was standardized between heifers, were obtained (Days 59 and 89).(ABSTRACT TRUNCATED AT 250 WORDS)