A functional analysis of the Tn5 transposase. Identification of domains required for DNA binding and multimerization

A series of deletions were constructed in the 476 amino acid Tn5 transposase in order to assemble an initial domain structure for this protein. The first four amino acids were found to be important for transposition activity but not for DNA binding to the Tn5 outside end (OE). Larger amino-terminal deletions result in the complete loss of transposition in vivo and the concomitant loss of specific DNA binding. Four point mutants and a six base-pair deletion in the amino terminus between residues 20 and 36 were also found to impair DNA binding to the OE. Analysis of a series of carboxy-terminal deletions has revealed that the carboxy terminus may actually mask the DNA binding domain, since deletions to residues 388 and 370 result in a large increase in DNA binding activity. In addition, the carboxy-terminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE complex indicative of a change in the oligomeric state of this complex. Further carboxy-terminal deletions beyond residue 370 also abolished DNA binding activity. These results indicate that the first four amino acids of Tnp are important for transposition but not DNA binding, a region between residues 5 and 36 is critical for DNA binding, the wild-type carboxy terminus acts to inhibit DNA binding, and that a region towards the carboxy terminus, defined by residues 370 to 387, is critical for Tnp multimeric interactions.     

Long-term changes in excitability induced by protein kinase C activation in Aplysia sensory neurons

Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.     

The impact of true partnership between a university Level I trauma center and a community Level II trauma center on patient transfer practices

OBJECTIVE: To examine the effect of a clinical and administrative partnership with an academic urban Level I trauma center on the patient transfer practices at a suburban/rural Level II center. METHODS: Data for 2 years before affiliation (PRE) abstracted from inpatient charts and the trauma registry were compared with that for 2 years after (POST). The following data were collected: number of, reason for, and destination and demographics of transfers. Chi(2) test and t test analyses were used; p < 0.05 defined significance; data are mean +/- SEM. RESULTS: Transfer rate increased from 4% PRE to 6.9% (p = 0.001) POST with no significant difference in age, Glasgow Coma Scale score, Injury Severity Score, or Revised Trauma Score. Repatriation occurred in 12.8% POST (none PRE). The current Level I facility accepted 1.8% of all transfers PRE and 36.4% POST (p = 0.0001). PRE/POST rates by reason are as follows: pediatric, 14.6%/9.0% (p = 0.04); intensive care unit, 0.4%/1.7% (p = 0.13); complex orthopedic, 100%/0% (p = 0.005); vascular, 50%/0% (p = 0.008); spinal cord injury, 100%/100%; and ophthalmologic, 0%/100% (p = 0.005). CONCLUSIONS: In this experience of Level I/II partnership (1) transfer patterns were altered, (2) select patient cohort transfers decreased (pediatric, complex orthopedic, vascular), whereas others increased (aortic work-up), and (3) repatriation rates were low.     

Protective effects of a recombinant N-terminal fragment of bactericidal/permeability increasing protein on endotoxic shock in conscious rabbits

Endotoxin (lipopolysaccharide, LPS) can induce shock, multiple organ failure, and death. A recombinant N-terminal fragment of bactericidal/permeability increasing protein, rBPI23, binds with high affinity to gram-negative bacterial LPS and neutralizes its biological activity. We sought to determine the effect of rBPI23 on LPS-induced respiratory dysfunction and cardiovascular depression in conscious rabbits. Rabbits were injected with Escherichia coli O113 LPS (6 micrograms/kg) and treated with rBPI23 (2 mg/kg), vehicle, or control protein after recovery from surgery performed to implant catheters for hemodynamic assessments and intravenous injections. LPS challenge caused respiratory dysfunction including tachypnea, significant decreases in arterial O2 tension (PO2), arterial oxygen content, and an increase in alveolar-arterial O2 gradient (A-aDO2). LPS administration also resulted in profound and prolonged decreases in mean arterial blood pressure and cardiac index. Treatment with rBPI23 prevented LPS-induced respiratory dysfunction and significantly ameliorated the cardiovascular depression. 5 of 16 LPS-challenged animals died of respiratory failure and acidosis, whereas none died in the rBPI23 treated group (p = .11). The results demonstrate that rBPI23 protects animals against LPS-induced cardiopulmonary depression in endotoxic shock.     

Fish oil supplementation with and without added vitamin E differentially modulates plasma antioxidant concentrations in healthy women

5-[4-(2-Carboxyethylcarbamoyl)phenylazo]salicylic acid disodium salt dihydrate (CAS 80573-04-2, BX661A) is developed as a therapeutic agent for ulcerative colitis. To clarify the mechanisms of action of BX661A, the effects of BX661A and its metabolites 5-aminosalicylic acid (5-ASA) and 4-aminobenzoyl-beta-aline (4-ABA) on polymorphonuclear (PMN) leukocyte chemotaxis and production of reactive oxygen species (ROS) from PMN cells were investigated and compared with the effects of 2-hydroxy-5-[[4-[(2-pyridinylamino)sulfonyl]phenyl]azo]-benzoic acid (CAS 599-79-1, SASP) and its metabolite 4-amino-N-2-pyridinyl-benzenesulfonamide (CAS 144-83-2, SP). 1. BX661A, SASP and SP concentration-dependently inhibited guinea pig PMN cell chemotaxis induced by zymosan-activated serum (IC50 = 1.39, 2.17 mmol/l, respectively) and by N-formyl-methionyl-leucyl-phenylalanine (FMLP) with IC50 values of 0.55, 0.06 and 0.66 mmol/l, respectively. 5-ASA and 4-ABA weakly affected the PMN cell chemotaxis induced by zymosan-activated serum (both IC50 values > or = 10 mmol/l) and by FMLP (IC50 > or = 10 and 8.05 mmol/l, respectively). 2. BX661A, SASP and SP concentration-dependently inhibited human PMN cell chemotaxis induced by FMLP with IC50 values of 0.68, 0.05 and 2.68 mmol/l, respectively, but both IC50 values of 5-ASA and 4-ABA were > 10 mmol/l. 3. BX661A, SASP, 5-ASA, 4-ABA and SP inhibited ROS production from rat PMN cells stimulated by FMLP in a concentration-dependent manner (IC50 = 58.4, 27.5, 0.61, 1242 and 13.9 mmol/l, respectively). 4. BX661A, SASP, 5-ASA, 4-ABA and SP inhibited ROS production from human PMN cells stimulated by FMLP in a concentration-dependent manner (IC50 = 67.4, 46.1, 0.69, 748 and 8.31 mumol/l, respectively). These results suggest that BX661A itself has inhibitory effects against PMN cell chemotaxis and ROS production from PMNs and that 5-ASA, which is the active moiety of BX661A, has a potent inhibitory effect against ROS production from PMNs. Therefore, these effects may be partially involved in the therapeutic effects of BX661A on ulcerative colitis.     

Nutrient antioxidants in gastrointestinal diseases

Oxidative stress appears to play a role in the pathogenesis of a number of gastrointestinal disease states, including pancreatitis; gastric and duodenal ulcer disease; IBD; gastric, esophageal, and colon cancers; and hepatic injury secondary to alcohol, metal storage disorders, hepatitis, and ischemia/reperfusion injury. The nutritional antioxidants are attractive potential therapeutic and chemopreventive agents because they are inexpensive and have a relatively low toxicity profile. A word of caution should be noted: Some antioxidants, such as vitamin C, can be prooxidant under certain conditions, and systemically altering the redox state may have untoward effects on the inflammatory response in certain disease states. Thus, at the current time, antioxidant therapy should be restricted to randomized, controlled clinical trials, in which treatment effects can be closely monitored, and therapeutic efficacy can be determined with scientific accuracy.     

Correlation of in vitro susceptibility testing results for amoxicillin-clavulanate and ampicillin-sulbactam using a panel of beta-lactamase-producing Enterobacteriaceae

Laryngospasm occurring after tracheal extubation in children is potentially dangerous. This study uses acupuncture with bloodletting at the Shao Shang (L 11) or Shang Yang (Li 1) acupoints to investigate whether this technique can prevent or treat laryngospasm. Seventy-six patients were randomly divided into two groups. Patients in the acupuncture group (n = 38) were treated with bilateral Shao Shang acupunctures at the end of the operation. Patients in the control group (n = 38) were not. The incidence of laryngospasm in the acupuncture group (5.3%) was less than that in the control group (23.7%) (p < 0.05). If laryngospasm developed, patients were immediately treated with acupuncture at either the Shao Shang or Shang Yang acupoints. As judged by an increase in peripheral oxygen saturation, the laryngospasm was relieved within 1 min of acupuncture in all patients. It is concluded that acupuncture with bloodletting at the Shao Shang acupoint may prevent and treat laryngospasm occurring after tracheal extubation in children.     

Inhibitory action of YJA20379, a new proton pump inhibitor on Helicobacter pylori growth and urease

The use of an episiotomy for vaginal delivery is a controversial topic in modern obstetrics. If one is done, however, correct technique of perineal trauma repair is important. The usual episiotomy has traditionally been closed with interrupted suture. The use of a less reactive material, e.g. polyglycolic acid (Dexon), seem to be suitable for subcutaneous skin closure and beneficial in terms of acute postpartum discomfort and healing. The comparison of healing and patient comfort parameters between interrupted and subcutis polyglycolic acid suture used for episiotomy repair after delivery was done. Patients had follow-up during hospital stay, and two months after delivery a self administered questionnaire was sent to all women who participated, enquiring about perineal pain, resumption of sexual intercourse and cosmetics of suture line. Of 52 patients who had repair with interrupted suture, 21 were lost to follow-up. Of 65 gravidas who had repair with subcutis suture, 23 were lost to follow-up. At the 3rd day postpartum examination, patients with subcutis sutures had significantly better healing. An inflammatory process was present in 2 of 52 patients with interrupted sutures comparable with 1 of 65 in the subcutis group, and a gaping wound in 0 of 52 and 1 of 65, respectively. Recovery of function, measured by resumption of sexual activity by 8 weeks, was demonstrated in 5 of 31 patients with interrupted sutures versus 24 of 42 patients who had subcutis sutures. Episiotomy repair with subcutis polyglycolic acid (Dexon) offers significant advantages over traditional interrupted suture, both in terms of wound healing and resumption of sexual activity.