An Examination of the Differential Sensitivity to Ketolide Antibiotics in <Emphasis Type="Italic">ermB</Emphasis> Strains of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC₅₀ values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC₅₀ values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC₅₀ values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.     

Larval development of Phoronis pallida (Phoronida): implications for morphological convergence and divergence among larval body plans

Morphological variation among larval body plans must be placed into a phylogenetic and ecological context to assess whether similar morphologies are the result of phylogenetic constraints or convergent selective pressures. Investigations are needed of the diverse larval forms within the Lophotrochozoa, especially the larvae of phoronids and brachiopods. The actinotroch larva of Phoronis pallida (Phoronida) was reared in the laboratory to metamorphic competence. Larval development and growth were followed with video microscopy, SEM, and confocal microscopy. Early developmental features were similar to other phoronid species. Gastrulation was accomplished by embolic invagination of the vegetal hemisphere. Mesenchymal cells were found in the remaining blastocoelic space after invagination began. Mesenchymal cells formed the body wall musculature during the differentiation of larval features. Body wall musculature served as the framework from which all other larval muscles proliferated. Larval growth correlated best with developmental stage rather than age. Consistent with other phoronid species, differentiation of juvenile tissues occurred most rapidly at the latest stages of larval development. The minimum precompetency period of P. pallida was estimated to be approximately 4-6 weeks. Previously published studies have documented that the planktonic embryos of P. pallida develop faster than the brooded embryos of P. vancouverensis. However, these data showed that the difference in developmental rate between the two species decreased in succeeding larval stages. There may be convergent selective pressures that result in similar timing to metamorphic competence among phoronid and brachiopod planktotrophic larval types. Morphological differences between these larval types result from heterochronic developmental shifts in the differentiation of juvenile tissue. Similarities in the larval morphology of phoronids and basal deuterostomes are likely the result of functional and developmental constraints rather than a shared (recent) evolutionary origin. These constraints are imposed by the functional design of embryological stages, feeding structures, and swimming structures.     

Biphasic modulation of vascular nitric oxide catabolism by oxygen

Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vascular tone. Lack of NO bioavailability can result in cardiovascular disease. NO bioavailability is determined by its rates of generation and catabolism; however, it is not known how the NO catabolism rate is regulated in the vascular wall under normoxic, hypoxic, and anaerobic conditions. To investigate NO catabolism under different oxygen concentrations, studies of NO and O2 consumption by the isolated rat aorta were performed using electrochemical sensors. Under normoxic conditions, the rate of NO consumption in solution was enhanced in the presence of the rat aorta. Under hypoxic conditions, NO consumption decreased in parallel with the O2 concentration. Like the inhibition of mitochondrial respiration by NO, the inhibitory effects of NO on aortic O2 consumption increased as O2 concentration decreased. Under anaerobic conditions, however, a paradoxical reacceleration of NO consumption occurred. This increased anaerobic NO consumption was inhibited by the cytochrome c oxidase inhibitor NaCN but not by the free iron chelator deferoxamine, the flavoprotein inhibitor diphenylene iodonium (10 microM), or superoxide dismutase (200 U/ml). The effect of O2 on the NO consumption could be reproduced by purified cytochrome c oxidase (CcO), implying that CcO is involved in aortic NO catabolism. This reduced NO catabolism at low O2 tensions supports the maintenance of effective NO levels in the vascular wall, reducing the resistance of blood vessels. The increased anaerobic NO catabolism may be important for removing excess NO accumulation in ischemic tissues.     

Blood pressure regulates the activity and function of the Na-K-2Cl cotransporter in vascular smooth muscle

The Na-K-2Cl cotransporter (NKCC1) is one of several transporters that have been linked to hypertension, and its inhibition reduces vascular smooth muscle tone and blood pressure. NKCC1 in the rat aorta is stimulated by vasoconstrictors and inhibited by nitrovasodilators, and this is linked to the contractile state of the smooth muscle. To determine whether blood pressure also regulates NKCC1, we examined the acute effect of hypertension on NKCC1 in rats after aortic coarctation. In the hypertensive aorta (28-mmHg rise in mean blood pressure), an increase in NKCC1 activity (measured as bumetanide-sensitive (86)Rb efflux) was apparent by 16 h and reached a plateau of 62% greater than control at 48 h. In contrast, there was a slight decrease in NKCC1 activity in the hypotensive aorta (21% decrease in mean blood pressure). Measurement of NKCC1 mRNA by real-time PCR revealed a fivefold increase in the hypertensive aorta compared with the hypotensive aorta or sham aorta. The inhibition by bumetanide of isometric force response to phenylephrine was significantly greater in the hypertensive aorta than in the control aorta or hypotensive aorta. We conclude that NKCC1 in rat aortic smooth muscle is regulated by blood pressure, most likely through changes in transporter abundance. This upregulation of NKCC1 is associated with a greater contribution to force generation in the hypertensive aorta. This is the first demonstration that NKCC1 in vascular smooth muscle is regulated by blood pressure and indicates that this transporter is important in the acute response of vascular smooth muscle to hypertension.     

Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity

c-Jun NH₂-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle. electroporation; gene delivery; muscle contraction; exercise     

Locomotion induced by non-contingent intracranial electrical stimulation: Dopamine dependence and general characteristics

Intracranial self-stimulation (ICSS) is induced by delivery of electrical stimulation contingent upon a response such as bar pressing. This procedure has been widely used to investigate the brain reward system. Recent investigations, however, have noted that non-contingent electrical stimulation, also called experimenter applied stimulation (EAS), produces a unique set of locomotion behaviors that appear to be related to ICSS, and that these behaviors resemble locomotion similar to those elicited by dopamine enhancing drugs. However, little is known about the general characteristics of EAS-induced locomotion. While ICSS appears to be robust, long lasting, and highly rewarding in that the rat will invest vast amounts of time or energy to obtain the electrical stimulation, these parameters have not been explored for EAS. Moreover, the dopamine dependence of EAS-evoked locomotion is also not firmly established. Thus, the present study investigated dopamine dependence and general characteristics of the EAS-induced locomotion to determine its similarity to ICSS. Results suggested that motor and limbic systems were strongly activated by non-contingent EAS, and that the resulting locomotion was dopamine dependent, robust, continued across long time horizons, and was greater than that evoked by contingent electrical stimulation.     

Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.     

The serratus anterior subslip: anatomy and implications for facial and hand reanimation

The ideal donor muscle for facial and hand reanimation has yet to be found. Donor muscles commonly used today, such as the gracilis and pectoralis minor, are limited by bulkiness and the number of force vectors they can provide. In the authors' study of 50 fresh cadaver serratus anterior muscles, they further describe neurovascular anatomy of the muscle slip (i.e., the portion of the muscle that inserts on a rib) and subslip (superficial or deep subdivision of the slip after division along a loose areolar plane). All 260 slips could be separated into a deep and a superficial subslip, yielding a total of 520 subslips. A branch of the serratus artery (a terminal branch of the thoracodorsal artery serving the lower five to seven slips of the muscle) and a branch of the long thoracic nerve were identified for each of these. Deep subslips were thinner than superficial subslips, both at the origin of the slip on the rib periosteum (2.4 mm versus 3.0 mm, p < 0.0001) and centrally at the serratus artery (3.3 mm versus 4.0 mm, p < 0.0001). In addition, the subslips of the most inferior slip were thinner than those of more superior slips, both at the origin of the slip (2.3 mm versus 2.8 mm, p < 0.0001) and at the serratus artery (3.0 mm versus 3.8 mm, p < 0.0001). Fine anastomosing vessels were present between the slips and the subslips. The average number of anastomosing vessels present between adjacent slips was 1.7, and 2.1 anastomosing vessels were present between the subslips of a given slip. Given the thinness of these vessels (all less than 0.2 mm) compared with those of the vascular pedicle of the subslip (mean, 0.7 mm; all greater than 0.4 mm), the authors believe these can be safely divided without compromising subslip vascularity. After division of these vessels, a mean length of 9.6 +/- 1.5 cm is available to allow independent orientation of each subslip. When the serratus muscle flap is separated into its component subslips, a maximum of 10 possible force vectors may be transferred on a single vascular pedicle. Subslips are significantly thinner than donor muscles commonly used today. These two advantages offer the potential for significant functional and aesthetic improvement when the serratus anterior muscle flap is used for face and hand reanimation. Mimetic muscles such as the orbicularis oculi and orbicularis oris could possibly be reconstructed in their proper anatomical positions.