Determination of skin concentrations of enrofloxacin in dogs with pyoderma

OBJECTIVE: To compare serum and skin concentrations of enrofloxacin in dogs with pyoderma with those of clinically normal dogs and to evaluate concentrations in dogs with superficial versus deep pyoderma. ANIMALS: 16 clinically normal dogs and 16 dogs with pyoderma. PROCEDURE: Enrofloxacin (approx 5 mg/kg of body weight, PO) was administered daily to all dogs. Serum samples and skin biopsy specimens were obtained on day 1 at 3 hours after drug administration and on day 3 immediately before and 3 hours after drug administration. Samples and specimens were assayed by high-performance liquid chromatography. Morphometric analysis was performed on skin biopsy specimens to determine correlation between inflammatory cells and peak tissue enrofloxacin concentration on day 1. RESULTS: Morphometric analysis revealed high correlation between dermal inflammatory cell count and drug concentration in dogs with pyoderma. CONCLUSIONS: At mean dosage of 5 mg/kg once daily, enrofloxacin tissue concentrations were significantly greater in dogs with pyoderma at 3 hours after pill administration. Enrofloxacin tissue concentration on day 3 at 3 hours after pill administration was 12.4 times the 90% minimum inhibitory concentration of enrofloxacin for Staphylococcus intermedius. CLINICAL RELEVANCE: In dogs with pyoderma, therapeutic tissue concentrations of enrofloxacin are reached as early as 3 hours after drug administration.     

Cloning and expression of chicken CLIP-170 and restin isoforms

Isomeric norbornane-derived rigid analogs mimicking different potential conformations of ACPD (1-aminocyclopentane-1,3-dicarboxylic acid) and glutamic acid have been synthesized, via the hydantoin route, to be used as conformational probes for bioactive conformations at the glutamatergic receptors of the central nervous system. Activities on metabotropic receptors mGluR1 and mGluR2 are reported and discussed.     

The role of the GLUT 4 transporter in regulating rat myoblast glucose transport processes

Movement of the malaria parasite into a host erythrocyte during invasion is thought to involve polymerization of parasite actin. We have used F-actin affinity chromatography to isolate actin-binding proteins from Plasmodium knowlesi merozoites, in an attempt to identify proteins responsible for regulating parasite actin polymerization during invasion. Five major proteins, of molecular masses 75, 70, 48, 40 and 34 kDa, were reproducibly eluted from the F-actin columns. The 70 kDa actin-binding protein was identified by tryptic peptide microsequencing as heat shock protein-70 kDa (HSC70); this identification was confirmed by Western blotting with anti-HSC70 antibody, and binding of the protein to ATP-agarose. A doublet of 32/34-kDa proteins coeluted with parasite HSC70 from the F-actin and ATP-agarose columns; a complex of these three proteins was also observed by gel filtration chromatography Highly enriched fractions containing the Plasmodium HSC70/32/34 complex inhibited the polymerization of rabbit skeletal muscle actin, in vitro. This capping activity was calcium-independent, and abrogated by phosphatidylinositol 4,5-bisphosphate. The average length of the actin filaments polymerized in presence of the HSC70/32/34-kDa complex was significantly shorter than in the absence of the complex, consistent with a capping activity. The capping or uncapping of actin filament ends by the HSC70/32/34-kDa complex during invasion could provide a mechanism for localized actin filament growth and movement of the parasite into the host cell.     

Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.     

Impact of a clinical pathway for elective infrarenal aortic reconstructions

OBJECTIVE: To determine the impact of a clinical pathway for elective infrarenal aortic reconstruction on outcome, resource utilization, and cost in a university medical center. SUMMARY BACKGROUND DATA: Clinical pathways have been reported to control costs, reduce resource utilization, and maintain or improve the quality of patient care, although their use during elective aortic reconstructions remains unresolved. METHODS: A clinical pathway was developed for elective infrarenal aortic reconstructions by a multidisciplinary group comprised of representatives from each involved service. The prepathway practice and costs were analyzed and an efficient, cost-effective practice with specific outcome measures was defined. The impact of the pathway was determined by retrospective comparison of outcome, resource utilization, and cost (total and direct variable) between the pathway patients (PATH, n = 45) and a prepathway control group (PRE, n = 20). RESULTS: There were no significant differences in the patient demographics, comorbid conditions, operative indications, or type of reconstruction between the groups. There were no operative deaths and the overall complication rate (PRE, 35% vs. PATH, 34%) was similar. The pathway resulted in significant decreases in the total length of stay and preoperative length of stay and a trend toward a significant decrease (p = 0.08) in the intensive care length of stay for the admission during which the operation was performed. The pathway also resulted in significant decreases in both direct variable and total hospital costs for this admission, as well as a significant decrease in the overall direct variable and total hospital costs for the operative admission and the preoperative evaluation (< or =30 days before operative admission). Despite these reductions, the discharge disposition, 30-day readmissions, and number of postoperative clinic visits within 90 days of discharge were not different. CONCLUSIONS: Implementation of a clinical pathway for elective infrarenal aortic reconstructions dramatically decreased resource utilization and hospital costs without affecting the quality of patient care and did not appear to shift the costs to another setting.     

Anatomy and physiology of principal cells of the medial nucleus of the trapezoid body (MNTB) of the cat

We have recorded from principal cells of the medial nucleus of the trapezoid body (MNTB) in the cat's superior olivary complex using either glass micropipettes filled with Neurobiotin or horseradish peroxidase for intracellular recording and subsequent labeling or extracellular metal microelectrodes relying on prepotentials and electrode location. Labeled principal cells had cell bodies that usually gave rise to one or two primary dendrites, which branched profusely in the vicinity of the cell. At the electron microscopic (EM) level, there was a dense synaptic terminal distribution on the cell body and proximal dendrites. Up to half the measured cell surface could be covered with excitatory terminals, whereas inhibitory terminals consistently covered about one-fifth. The distal dendrites were very sparsely innervated. The thick myelinated axon originated from the cell body and innervated nuclei exclusively in the ipsilateral auditory brain stem. These include the lateral superior olive (LSO), ventral nucleus of the lateral lemniscus, medial superior olive, dorsomedial and ventromedial periolivary nuclei, and the MNTB itself. At the EM level the myelinated collaterals gave rise to terminals that contained nonround vesicles and, in the LSO, were seen terminating on cell bodies and primary dendrites. Responses of MNTB cells were similar to their primary excitatory input, the globular bushy cell (GBC), in a number of ways. The spontaneous spike rate of MNTB cells with low characteristic frequencies (CFs) was low, whereas it tended to be higher for higher CF units. In response to short tones, a low frequency MNTB cell showed enhanced phase-locking abilities, relative to auditory nerve fibers. For cells with CFs >1 kHz, the short tone response often resembled the primary-like with notch response seen in many globular bushy cells, with a well-timed onset component. Exceptions to and variations of this standard response were also noted. When compared with GBCs with comparable CFs, the latency of the MNTB cell response was delayed slightly, as would be expected given the synapse interposed between the two cell types. Our data thus confirm that, in the cat, the MNTB receives and converts synaptic inputs from globular bushy cells into a reasonably accurate reproduction of the bushy cell spike response. This MNTB cell output then becomes an important inhibitory input to a number of ipsilateral auditory brain stem nuclei.     

alpha-latrotoxin action probed with recombinant toxin: receptors recruit alpha-latrotoxin but do not transduce an exocytotic signal

alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Ialpha. We have now produced recombinant alpha-latrotoxin (LtxWT) that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three alpha-latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor-binding affinities identical to wild-type LtxWT, bound to CL1 and neurexin Ialpha as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.     

Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 A resolution

BACKGROUND: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively. IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function. The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently. RESULTS: IF-5A from the archebacterium Pyrobaculum aerophilum has been heterologously expressed in Escherichia coli with selenomethionine substitution. The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 A. Unmodified P. aerophilum IF-5A is found to be a beta structure with two domains and three separate hydrophobic cores. CONCLUSIONS: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between beta strands beta4 and beta5; this lysine residue is freely solvent accessible. The C-terminal domain is found to be homologous to the cold-shock protein CspA of E. coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.