Purification and characterization of macrolide 2''-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Hyperimmune and high-titered polyclonal pneumococcal antisera, specific for cross-reactive types within groups, were produced in adult rabbits. Purified capsular polysaccharide was injected intravenously into adult rabbits. One week later, these rabbits were given multiple intravenous injections of formalin-inactivated pneumococci of the cross-reactive type by an established method. Each of the resultant antisera were specific for the cross-reactive type indicating that the previous injection of the polysaccharide had induced epitope-specific tolerance. This method was successful for production of antisera against pneumococcal types 6A, 6B, 9N, 9V, 19F and 19A. Polyclonal rabbit pneumococcal antisera have some advantages over murine monoclonal antibodies for serologic studies and this method should be applicable for producing type-specific antibodies to cross-reactive polysaccharides of clinical interest. Further, this method is simpler and generally produces higher titered monovalent (factor) reagents than absorbed antisera.     

A Highlights from MBoC Selection: Regulation of the formin Bnr1 by septins anda MARK/Par1-family septin-associated kinase

Formin-family proteins promote the assembly of linear actin filaments and are required to generate cellular actin structures, such as actin stress fibers and the cytokinetic actomyosin contractile ring. Many formin proteins are regulated by an autoinhibition mechanism involving intramolecular binding of a Diaphanous inhibitory domain and a Diaphanous autoregulatory domain. However, the activation mechanism for these Diaphanous-related formins (DRFs) is not completely understood. Although small GTPases play an important role in relieving autoinhibition, other factors likely contribute. Here we describe a requirement for the septin Shs1 and the septin-associated kinase Gin4 for the localization and in vivo activity of the budding yeast DRF Bnr1. In budding yeast strains in which the other formin, Bni1, is conditionally inactivated, the loss of Gin4 or Shs1 results in the loss of actin cables and cell death, similar to the loss of Bnr1. The defects in these strains can be suppressed by constitutive activation of Bnr1. Gin4 is involved in both the localization and activation of Bnr1, whereas the septin Shs1 is required for Bnr1 activation but not its localization. Gin4 promotes the activity of Bnr1 independently of the Gin4 kinase activity, and Gin4 lacking its kinase domain binds to the critical localization region of Bnr1. These data reveal novel regulatory links between the actin and septin cytoskeletons.     

A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.     

High-performance anion-exchange chromatography of ultraviolet-absorbing constituents of human urine

A 100-μl urine sample was chromatographed on a column packed with a strongly basic macroreticular anion-exchange resin (Diaion CDR-10, 5– μm diameter with a nominal 35% cross linkage). The elution was performed with a linear acetate gradient from 0 to 6.0 M at an average flow-rate of 0.72 ml/min and at an average pressure of 104 kg/cm². The relative standard deviation of retention times and peak height was ± 4% or less. The properties of the macroreticular anion-exchange resin, the effect of the particle size, the pH of acetate buffers, and the effect of the flow-rate of the eluent on the separation were investigated. Thirty three components of urine were then resolved and named.     

Identification of thiol group at the estrogen-binding site on the cytoplasmic receptor from rabbit uterus by affinity labeling

The interaction of estrogen with its receptor from rabbit uterus was studied using the affinity labeling technique. An affinity labeling reagent, 3-hydroxy-17β-(p-nitrophenyldithio)-1,3,5(10)-estratriene (Reagent A) was synthesized. The compound was designed to meet two requirements: (1) it binds specifically to the steroid-binding site of the receptor; (2) it has functional group capable of forming a covalent bond with an amino acid residue at or near the binding site. The first requirement was demonstrated by competitive binding assay and sedimentation analysis. If the binding affinity of 17β-estradiol is defined as 100, that of Reagent A is found to be 0.05. It was shown that the binding was specific for the estradiol receptor. The second requirement was examined by extraction of Reagent A with simple model compounds. 10 μM Reagent A gave rise to approx. 1.3 μM p-nitrothiophenol in the presence of a large excess of l-cysteine or reduced l-glutathione under the experimental conditions employed, indicating that a covalent bond was formed between Reagent A and the model compounds. Furthermore, I have presented evidence by using polyacrylamide gel electrophoresis that ³H-labeled Reagent A is linked covalently to the estrogen-binding site on the receptor of rabbit uterus. The experiment also fulfilled the first requirement. These results indicate that the thiol group present at the binding site is directly involved in the estradiol-receptor binding.     

Functional significance of manganese catalase in Lactobacillus plantarum.

A strain of Lactobacillus plantarum which was unable to produce manganese (Mn)catalase (ATCC 8014) grew somewhat more rapidly and to a slightly higher plateau density than did an Mn catalase-positive strain (ATCC 14421), and this was the case during aerobic or anaerobic growth. However, when maintenance of viability was measured during the stationary phase of the growth cycle, the advantage provided by Mn catalase was obvious. Thus, the viability of ATCC 14431 was undiminished over 21 h of aerobic incubation, during the stationary phase, whereas that of ATCC 8014 decreased by seven orders of magnitude. Addition of catalase to the medium or growth in the presence of hemin, which allows catalase synthesis, protected ATCC 8014 against this loss of viability. Suppression of Mn catalase within ATCC 14431 by treatment with NH2OH caused the cells to lose viability when exposed to 4 mM H2O2.     

Effects of temperature on basal and insulin-stimulated glucose transport activities in fat cells. Further support for the translocation hypothesis of insulin action

Effects of temperature on glucose transport in fat cells were studied. In this system, the basal (no insulin) glucose transport activity was higher at approximately 25-30 degrees C than at 37 degrees C, as previously reported (Vega, F. V., and Kono, T. (1979) Arch. Biochem. Biophys. 192, 120-127). The stimulatory effect of low temperature (or the insulin-like effect) was reversible and apparently required metabolic energy for both its forward and reverse reactions. By lowering the ATP level with 2,4-dinitrophenol, one could separately determine the insulin-like stimulatory effect of low temperature and its inhibitory effect on the transport process itself. The maximum level of stimulation by low temperature was greater at 10 degrees C than at 25-30 degrees C, but the rate of stimulation was considerably slower at 10 degrees C than at 25-30 degrees C. When cells were exposed to low temperature, the glucose transport activity in the plasma membrane-rich fraction was increased, while that in the Golgi-rich fraction was decreased. The Arrhenius plot of the basal glucose transport activity determined in the presence of dinitrophenol was apparently linear from 10 to 37 degrees C and parallel to that of the plus insulin activity measured either in the presence or absence of dinitrophenyl. Insulin itself slowly stimulated the glucose transport activity at 10 degrees C. These results are consistent with the view that (a) low temperature, like insulin, induces translocation of the glucose transport activity from an intracellular storage site to the plasma membrane, (b) insulin stimulates glucose transport activity without changing its activation energy, and (c) subcellular membranes do not entirely stop their movement at a low temperature, e.g, at 10 degrees C.     

Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation.

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.