ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin B17

In the maturation of the Escherichia coli antibiotic Microcin B17, the product of the mcbA gene is modified posttranslationally by the multimeric Microcin synthetase complex (composed of McbB, C, and D) to cyclize four Cys and four Ser residues to four thiazoles and four oxazoles, respectively. The purified synthetase shows an absolute requirement for ATP or GTP in peptide substrate heterocyclization, with GTP one-third as effective as ATP in initial rate studies. The ATPase/GTPase activity of the synthetase complex is conditional in that ADP or GDP formation requires the presence of substrate; noncyclizable versions of McbA bind to synthetase, but do not induce the NTPase activity. The stoichiometry of ATP hydrolysis and heterocycle formation is 5:1 for a substrate that contains two potential sites of modification. However, at high substrate concentrations (>50Km) heterocycle formation is inhibited, while ATPase activity occurs undiminished, consistent with uncoupling of NTP hydrolysis and heterocycle formation at high substrate concentrations. Sequence homology reveals that the McbD subunit has motifs reminiscent of the Walker B box in ATP utilizing enzymes and of motifs found in small G protein GTPases. Mutagenesis of three aspartates to alanine in these motifs (D132, D147, and D199) reduced Microcin B17 production in vivo and heterocycle formation in vitro, suggesting that the 45 kDa McbD has a regulated ATPase/GTPase domain in its N-terminal region necessary for peptide heterocyclization.     

The Coventry Award. Modular tibial insert micromotion. A concern with contemporary knee implants

This study investigates micromotion between modular tibial components, one of the causes of wear on the undersurface of polyethylene inserts. The authors measured motion at the interface of nine contemporary total knee implant designs by mechanically testing the implants in a servohydraulic testing machine. Anteroposterior and mediolateral motion between the tibial insert and baseplate were measured with an extensometer placed across the interface. These tests revealed that in all implants analyzed, sufficient motion occurred to create fretting at the modular interface. Although the testing configuration in this study was not a stimulation of in situ loading patterns, the authors observed hundreds of microns of motion even under a 100 N load and variability between implants of the same design, showing that there is room for improvement among locking mechanisms in modular total knee implants.     

Neonatal murine B lymphocytes respond to polysaccharide antigens in the presence of IL-1 and IL-6

Unlike adults, neonates are unable to respond to polysaccharide Ags, making them especially vulnerable to pathogenic encapsulated bacteria. Since the Ab response to polysaccharides in adult mice requires certain cytokines, it was hypothesized that neonatal murine B cells may be competent to respond to such Ags, but may fail to do so due to a deficiency of cytokines. Neonatal splenocyte cultures, which were otherwise unresponsive to trinitrophenyl (TNP)-Ficoll, a haptenated polysaccharide Ag, mounted an adult-like Ab response when supplemented with IL-1. However, IL-1 failed to induce such a response to TNP-Ficoll when purified B cells were used instead. Although IL-6 alone did not induce a response in whole spleen cells or purified B cells from neonates, it synergized with IL-1 in inducing purified neonatal B cells to respond to TNP-Ficoll. The avidity of the cytokine-induced neonatal anti-TNP Abs was comparable to that of Abs made by adult splenocyte cultures. One effect of IL-1 may be at the level of clonal expansion, since it induced neonatal B cells to proliferate in response to anti-IgM, which was further enhanced by IL-6. The spontaneous secretion of IL-1 by neonatal splenocytes was below the detection limit, while adult splenocytes secreted 30.8 +/- 5.2 U/ml, which is of the same order of magnitude as what was required to stimulate neonatal B cells to respond to TNP-Ficoll. Thus, the neonatal unresponsiveness to polysaccharide Ags could be due to the inability of a non-B cell population resident in the neonatal spleen to secrete sufficient quantities of IL-1.     

The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth

The Saccharomyces cerevisiae myosin-V, Myo2p, has been implicated in the polarized movement of several organelles and is essential for yeast viability. We have shown previously that Myo2p is required for the movement of a portion of the lysosome (vacuole) into the bud and consequently for proper inheritance of this organelle during cell division. Class V myosins have a globular carboxyl terminal tail domain that is proposed to mediate localization of the myosin, possibly through interaction with organelle-specific receptors. Here we describe a myo2 allele whose phenotypes support this hypothesis. vac15-1/myo2-2 has a single mutation in this globular tail domain, causing defects in vacuole movement and inheritance. Although a portion of wild-type Myo2p fractionates with the vacuole, the myo2-2 gene product does not. In addition, the mutant protein does not concentrate at sites of active growth, the predominant location of wild-type Myo2p. Although deletion of the tail domain is lethal, the myo2-2 gene product retains the essential functions of Myo2p. Moreover, myo2-2 does not cause the growth defects and lethal genetic interactions seen in myo2-66, a mutant defective in the actin-binding domain. These observations suggest that the myo2-2 mutation specifically disrupts interactions with selected myosin receptors, namely those on the vacuole membrane and those at sites of polarized growth.     

Topographic organization of connections between the hypothalamus and prefrontal cortex in the rhesus monkey

Prefrontal cortices have been implicated in autonomic function, but their role in this activity is not well understood. Orbital and medial prefrontal cortices receive input from cortical and subcortical structures associated with emotions. Thus, the prefrontal cortex may be an essential link for autonomic responses driven by emotions. Classic studies have demonstrated the existence of projections between prefrontal cortex and the hypothalamus, a central autonomic structure, but the topographic organization of these connections in the monkey has not been clearly established. We investigated the organization of bidirectional connections between these areas in the rhesus monkey by using tracer injections in orbital, medial, and lateral prefrontal areas. All prefrontal areas investigated received projections from the hypothalamus, originating mainly in the posterior hypothalamus. Differences in the topography of hypothalamic projection neurons were related to both the location and type of the target cortical area. Injections in lateral eulaminate prefrontal areas primarily labeled neurons in the posterior hypothalamus that were equally distributed in the lateral and medial hypothalamus. In contrast, injections in orbitofrontal and medial limbic cortices labeled neurons in the anterior and tuberal regions of the hypothalamus and in the posterior region. Projection neurons targeting orbital limbic cortices were more prevalent in the lateral part of the hypothalamus, whereas those targeting medial limbic cortices were more prevalent in the medial hypothalamus. In comparison to the ascending projections, descending projections from prefrontal cortex to the hypothalamus were highly specific, originating mostly from orbital and medial prefrontal cortices. The ascending and descending connections overlapped in the hypothalamus in areas that have autonomic functions. These results suggest that specific orbitofrontal and medial prefrontal areas exert a direct influence on the hypothalamus and may be important for the autonomic responses evoked by complex emotional situations.     

Effects of low radiation doses on Chinese hamster cells

The induction of cytogenetic damages after irradiation with single dose of gamma-rays (0.1-2 Gy) have been studied. It is shown non-linear curve for the induction of chromosome aberrations, detected by anaphase method. After irradiation in S-stage of the cell cycle at dose below 0.2 Gy the cells were more radiosensitive than after irradiation with doses 0.3-2 Gy. Between the phases of high radiosensitivity and radioresistance the reversal dose-effect relation was observed. This phenomenon was not marked for the cells after irradiation in G2-stage of the cell cycle. It is possible, this results could reflect an induced radioresistance at low dose of irradiation.     

Optical amplification of ligand-receptor binding using liquid crystals

Liquid crystals (LCs) were used to amplify and transduce receptor-mediated binding of proteins at surfaces into optical outputs. Spontaneously organized surfaces were designed so that protein molecules, upon binding to ligands hosted on these surfaces, triggered changes in the orientations of 1- to 20-micrometer-thick films of supported LCs, thus corresponding to a reorientation of approximately 10(5) to 10(6) mesogens per protein. Binding-induced changes in the intensity of light transmitted through the LC were easily seen with the naked eye and could be further amplified by using surfaces designed so that protein-ligand recognition causes twisted nematic LCs to untwist. This approach to the detection of ligand-receptor binding does not require labeling of the analyte, does not require the use of electroanalytical apparatus, provides a spatial resolution of micrometers, and is sufficiently simple that it may find use in biochemical assays and imaging of spatially resolved chemical libraries.     

Prevalence of multiple cardiovascular disease risk factors among women in the United States, 1992 and 1995: the Behavioral Risk Factor Surveillance System

We sought to examine the prevalence of self-reported multiple cardiovascular disease (CVD) risk factors (hypertension, high blood cholesterol, diabetes, overweight, and current smoking) among women in 1992 and 1995 in the United States using data from the Behavioral Risk Factor Surveillance System. In 1992, 37.5%, 34.4%, and 28.1% of women had zero, one, and two or more of the five risk factors, respectively. In 1995, the respective estimates were 35.5%, 34.3%, and 30%. In both years, the prevalence of two or more risk factors increased with age, decreased with educational level, was higher among black women (lowest among Hispanic women and women of other ethnic groups), and higher among women reporting cost as a barrier to healthcare. The percentage of women with two or more risk factors was higher in 1995 than in 1992 for 35 of 48 states, being statistically significant for 7 states. The percentage of women with at least two risk factors was not significantly lower in 1995 than in 1992 for any state. A higher percentage of women reported having multiple CVD risk factors in 1995 compared with 1992. A multifactorial approach to primary prevention and risk factor reduction should be encouraged to help reduce the prevalence and burden of CVD among women.