Purification and folding of recombinant bovine oxoglutarate/malate carrier by immobilized metal-ion affinity chromatography

A major obstacle to investigating the structure of membrane proteins is the difficulty in obtaining sufficient amounts of functional protein. The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been cloned as a fusion protein containing a C-terminal hexa-histidine tag. This fusion protein has been expressed at an abundant level in a mutant strain of Escherichia coli BL21 (DE3) called C41 (DE3). The protein accumulated as inclusion bodies and none was detected in the bacterial inner membrane. The denatured protein was refolded to reconstitute functional properties similar to the native protein. Solubilized inclusion body protein was immobilized using nickel-chelating affinity chromatography, and purified and refolded in a single step. The protein eluted as a monomer which was stable in mild detergent, at a yield equivalent to 15 mg active protein/liter bacterial culture. The reconstituted fusion protein displayed the same transport characteristics as the wild-type, demonstrating that the tag does not perturb the structure of the protein. The oxoglutarate carrier is one member of an extensive family of mitochondrial transport proteins. These proteins transport many different metabolites across the inner mitochondrial membrane and share a common mechanism of action. Therefore, it is likely that this folding protocol can be applied successfully to other mitochondrial transport proteins, thus providing sufficient protein for extensive crystallization trials with a wide range of family members.     

Inhibition of in vitro fertilization by mouse anti-mouse sperm sera and preliminary antigen identification

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.     

Streptomycin-Induced Phenotypic Suppression of Hydroxylamine-Induced Nonsense Mutants in the rII A Cistron of Bacteriophage T4

Twenty-one hydroxylamine-induced rII A cistron nonsense mutants were tested for streptomycin (SM)-induced phenotypic suppression by exposing Escherichia coli SBO (nonpermissive host) to phage in the presence and absence of SM. All nine amber, four of six ochre, and five of six opal mutants were phenotypically suppressible by SM. For suppressible mutants, the ratio of the average burst size in the presence of SM to size in the absence of SM ranged from 12 to 242 for the ambers, 3 to 33 for the ochres, and 4 to 14 for the opals. Increased susceptibility of the amber mutants to SM-induced phenotypic suppression relative to the susceptibility of the opal and ochre mutants may reflect a neighboring base effect, such that a 3′-terminal adenine inhibits misreading of a 5′-terminal uracil.     

The '3Rs' model and the concept of alternatives in animal research: a questionnaire survey

It has been 45 years since Russell and Burch first proposed the concept of the '3Rs', yet it remains unclear how those individuals involved in animal research view and implement these concepts. The authors used a questionnaire survey to determine how well-known experts judged issues related to the 3Rs.     

Macromolecular adducts of ethylene oxide: a literature review and a time-course study on the formation of 7-(2-hydroxyethyl) guanine following exposures of rats by inhalation

The results of efforts to identify and quantify macromolecular adducts of ethylene oxide (ETO), to determine the source and significance of background levels of these adducts, and to generate molecular dosimetry data on these adducts are reviewed. A time-course study was conducted to investigate the formation and persistence of 7-(2-hydroxyethyl)guanine (7-HEG; Fig. 1) in various tissues of rats exposed to ETO by inhalation, providing information necessary for designing investigations on the molecular dosimetry of adducts of ETO. Male F344 rats were exposed 6 h/day for up to 4 weeks (5 days/wk) to 300 ppm ETO by inhalation. Another set of rats was exposed for 4 weeks to 300 ppm ETO, and then killed 1–10 days after cessation of exposures. DNA samples from control and treated rats were analyzed for 7-HEG using neutral thermal hydrolysis, HPLC separation, and fluorescence detection. The adduct was detectable in all tissues of treated rats following 1 day of ETO exposure and increased approximately linearly for 3–5 days before the rate of increase began to level off. Concentrations of 7-HEG were greatest in brain, but the extent of formation was similar in all tissues studied. The adduct disappeared slowly from DNA, with an apparent half-life approx. 7 days. The shape of the formation curve and the in vivo half-life indicate that 7-HEG will approach steady-state concentrations in rat DNA by 28 days of ETO exposure. The similarity in 7-HEG formation in target and nontarget tissues indicates that the tissue specificity for tumor induction is due to factors in addition to DNA-adduct formation.     

Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Igh ^p.Abbreviations used in this paper Igh immunoglobulin heavy chain - SDS sodium dodecyl sulfate     

Central mechanisms of acute ANG II modulation of arterial baroreflex control of renal sympathetic nerve activity

Short-term intravenous infusion of angiotensin II (ANG II) into conscious rabbits reduces the range of renal sympathetic nerve activity (RSNA) by attenuating reflex disinhibition of RSNA. This action of ANG II to attenuate the arterial baroreflex range is exaggerated when ANG II is directed into the vertebral circulation, which suggests a mechanism involving the central nervous system. Because an intact area postrema (AP) is required for ANG II to attenuate arterial baroreflex-mediated bradycardia and is also required for maintenance of ANG II-dependent hypertension, we hypothesized that attenuation of maximum RSNA during infusion of ANG II involves the AP. In conscious AP-lesioned (APX) and AP-intact rabbits, we compared the effect of a 5-min intravenous infusion of ANG II (10 and 20 ng x kg(-1) x min(-1)) on the relationship between mean arterial blood pressure (MAP) and RSNA. Intravenous infusion of ANG II into AP-intact rabbits resulted in a dose-related attenuation of maximum RSNA observed at low MAP. In contrast, ANG II had no effect on maximum RSNA in APX rabbits. To further localize the central site of ANG II action, its effect on the arterial baroreflex was assessed after a midcollicular decerebration. Decerebration did not alter arterial baroreflex control of RSNA compared with the control state, but as in APX, ANG II did not attenuate the maximum RSNA observed at low MAP. The results of this study indicate that central actions of peripheral ANG II to attenuate reflex disinhibition of RSNA not only involve the AP, but may also involve a neural interaction rostral to the level of decerebration.     

Ontogeny of hemoglobins: Evidence for hemoglobin M

A new autosomal codominant hemoglobin mutation alters hemoglobin M of the primitive red cell line and hemoglobin D found in definitive cells. That Hb M and Hb D are altered by the same gene mutation supports the idea that Hb M shares a polypeptide chain with Hb D. It is concluded that in the switch from primitive hemoglobins to those of the definitive type, there are at least two α chains conserved; α^A of Hb E in Hb A and α^D of Hb M in Hb D.     

Cyclitol production in transgenic tobacco

High levels of cyclic sugar alcohols (cyclitols) correlate with tolerance to osmotic stress in a number of plant species. A gene encoding a cyclitol biosynthesis enzyme from a halophyte, Mesembryanthemum crystallinum has been introduced into tobacco. The gene, lmt1 , encodes a myo -inositol O -methyl transferase that, in M. crystallinum , catalyzes the first step in the stress-induced accumulation of the cyclitol pinitol. Tobacco transformed with the lmt1 cDNA under the control of the CaMV 35S promoter appeared phenotypically normal and exhibited IMT1 enzyme activity. Transformants accumulated a carbohydrate product not detectable in non-transformed control plants. This product was identified by HPLC and NMR as ononitol (1- d -4- O -methyl myo -inositol). Ononitol was a major carbohydrate constituent in leaf tissue of plants expressing the lmt1 gene, accumulating to up to 25% the level of sucrose in transformant seedlings. The identification of ononitol as the IMT1 product and the specific accumulation of this compound in transformed tobacco support a role for ononitol as a stable intermediate in pinitol biosynthesis and indicate that an epimerization activity lacking in tobacco is responsible for the conversion of ononitol to pinitol in M. crystallinum . The production of ononitol in tobacco indicates that plant carbohydrate metabolism is flexible and can accommodate the synthesis and accumulation of non-endogenous metabolites. The transgenic system described here will serve as a useful model to test the ability of cyclitols such as ononitol to confer tolerance to environmental stress in a normally glycophytic plant.