Affinity chromatography on anti-B1 monoclonal gels for purification and characterization of protein B1 from Escherichia coli ribonucleotide reductase

Affinity gels were prepared from four monoclonal antibodies against the B1 protein of ribonucleotide reductase of Escherichia coli. The gels were used to purify protein B1 and also to study some of its properties. Gels from the nonneutralizing monoclonal anti-B1-k bound as much as 2 mg of B1/mL and were employed to prepare essentially pure B1 protein in a single step from extracts of wild-type E. coli and strains overproducing the subunit. However, B1 prepared from wild-type extracts had a lowered specific activity, suggesting some denaturation during elution of the protein from the column. Addition of the allosteric effector dATP during affinity chromatography changed the chromatographic pattern. Some protein B2, the second subunit of the reductase, remained in all cases bound to the gels together with B1. The gel prepared from anti-B1-c retained two additional proteins. In other experiments involving binding of proteolytic fragments of B1 to various antibodies, we also found a striking effect of dATP, suggesting that dATP made protein B1 less accessible to proteolysis. In these experiments fragments around 15K still had the ability to bind monoclonals, making possible more detailed investigations of the structural contacts between B1 and the monoclonals.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

Studies on the mid-gut of the desert locust Schistocerca gregaria. II. Ultrastructure of the muscle coat and its innervation

The fine structure of the mid-gut musculature of the desert locust, Schistocerca gregaria is described and compared with that of the visceral muscles of other species. The gross morphology and fine structure of the nervous system which supplies the mid-gut muscle fibres is described.     

Effects of antihypertensive drugs on hepatic heme biosynthesis,and evaluation of ferrochelatase inhibitors to simplify testing of drugs for heme pathway induction

Effects of a series of antihypertensive drugs on the activity of δ-aminolevulinate synthase and on the formation of porphyrins and cytochrome P-450 were examined in the 18-day-old chick embryo liver in ovo. Hydralazine, pargyline, phenoxybenzamine, clonidine, and spironolactone were found to induce δ-aminolevulinate synthase in this system. These drugs therfore have the potential to precipitate clinical expression in human hereditary hepatic porphyrias and should be avoided or used with caution in patients with these disorders. Differential effects of these and other drugs were observed in the avian liver, in that δ-aminolevulinate synthase was more commonly induced thatn were porphyrins and cytochrome -450; the synthase was usually highest 6–12 h after injection, whereas porphyrins and cytochrome P-450 were highest at 24 h. Furthermore marked porphyrin accumulation was not seen with many drugs that induce σ-aminolevulinate synthase and cytochrome P-450 but was more characteristic of compounds that reduced the metabolism of protoporphyrin to heme, such as 1,4-dihydro-3,5-dicarbethoxycollidne (DDC) and high dose of hydralazine. A sensitive and convenient method to test for capacity to induce heme biosynthesis was adapted for use in the chick embryo liver. This employed a relatively small “priming” dose (0.25 mg) of DDC given with a drug being tested and a fluorometric assay of porphyrins in a liver homogenate obtained at 24 h. This simple method should facilitate screening for those drugs which induce the synthesis of δ-aminolevulinate synthase and/or cytochrome P-450 and are potentially dangerous to patients with hereditary hepatic porphyria.     

Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.

Structural maintenance of chromosomes (SMC) proteins play fundamental roles in higher-order chromosome dynamics from bacteria to humans. It has been proposed that the Bacillus subtilis SMC (BsSMC) homodimer is composed of two anti-parallel coiled-coil arms, each having an ATP-binding domain at its distal end. It remains totally unknown, however, how the two-armed structure supports ATP-dependent actions of BsSMC. By constructing a number of mutant derivatives including 'single-armed' BsSMC, we show here that the central hinge domain provides a structural flexibility that allows opening and closing of the two arms. This unique structure brings about bimodal regulation of the SMC ATPase cycle. Closing the arm can trigger ATP hydrolysis by allowing an end-end interaction within a dimer (intramolecular mode). When bound to DNA, ATP promotes a dimer-dimer interaction, which in turn activates their DNA-dependent ATPase activity (intermolecular mode). Our results reveal a novel mechanism of ATPase regulation and provide mechanistic insights into how eukaryotic SMC protein complexes could mediate diverse chromosomal functions, such as chromosome condensation and sister chromatid cohesion.     

Varieties of Anomalous Experience: Examining the Scientific Evidence

Varieties of Anomalous Experience: Examining the Scientific Evidence. Etzel Cardeña. Steven Jay Lynn. and Stanley Krippner. eds. Washington, DC: American Psychological Association, 2000. 476 pp.     

Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61 degrees N to 54 degrees S.

The effects of irradiances of 920 and 1200 mW m(-2) (biologically effective weighted irradiance) were examined in 2 Metarhizium album strains, 26 M. anisopliae strains, 1 M. flavoviride strain, and 1 M. taii strain isolated from sites located at latitudes from 61 degrees N to 54 degrees S. Conidia were exposed to UV-B from 1 to 6 h and subsequently examined for relative percentage culturability. Total dosage received at the end of the exposure periods ranged from 3.3 to 19.9 kJ m(-2) for the lower irradiance and from 4.3 to 25.9 kJ m(-2) for the higher irradiance. Both the irradiance values and the doses are environmentally realistic and can be observed even in temperate regions. The relationships between latitude of origin and UV-B tolerance were compared for the two levels of irradiance for the data from 1 and 2 h exposure. Exposure to both irradiances drastically reduced the relative percentage culturability of all strains. Tolerance to UV-B varied widely among strains and high variation was observed for both irradiances after all periods of exposure. After 1 h of exposure, a difference between the two irradiance levels was detectable, and this difference was magnified at longer irradiations. A significant quadratic relationship of decreasing UV-B tolerance with increasing latitude was observed after exposure of 1 and 2 h. The shape of the relationship did not differ for the two levels of irradiance. Also, we studied the effect of 1200 mW m(-2) irradiance on conidial germination time in 1 M. album strain, 7 M. anisopliae strains, and 1 M. taii strain. Exposure to UV-B delayed the germination of surviving conidia of all strains. In general, the delay in germination was directly proportional to the dose.     

The effects of Eleutherococcus senticosus and Panax ginseng on steroidal hormone indices of stress and lymphocyte subset numbers in endurance athletes.

A clinical trial was undertaken to investigate the effects of Eleutherococcus senticosus (ES) and Panax ginseng (PG) on competitive club-level endurance athletes engaged in their normal in-season training. Participants were matched for training stress and received a 33% ethanolic extract (8 mL/day) containing either ES, PG (equivalent to 4 g and 2 g/day of dried root, respectively), or a placebo. A pre-test and post-test were used to evaluate the effects of six weeks of supplementation on cortisol, testosterone, and testosterone to cortisol ratio (TCR) as well as circulating numbers of total T-cells, T-helper cells (CD4), T-suppressor cells (CD8), CD4 to CD8 ratio, natural killer cells, and B lymphocytes. None of the immune system variables changed significantly nor showed any clear trend from pre to post test in any of the treatment groups. No significant change in testosterone, cortisol or TCR was observed in the PG group. In the ES group, however, TCR decreased by 28.7% from 0.0464 to 0.0331 (P=0.03). The main contribution to this decrease appeared to be a non-significant (P= 0.07) 31% trend towards increased cortisol rather than a very small non-significant (P = 0.36) 7% decrease in the calculated mean for testosterone. This result suggested that contrary to initial expectation, ES increased rather than decreased hormonal indices of stress, which may be consistent with animal research suggesting a threshold of stress below which ES increases the stress response and above which ES decreases the stress response.