Effect of the general anesthetic halothane on the activity of the transverse tubule Ca(2+)-activated K+ channel

The effect of cycloheximide on increased splanchnic prostacyclin release following acute hemorrhage was studied in the rat. Male Sprague-Dawley rats were anesthetized and subjected to acute hemorrhage to 30 mm Hg for 30 min (shock) or sham shock. The superior mesenteric artery was cannulated and removed with its end organ intestine (SV + SI preparation) and perfused in vitro with oxygenated Krebs-Henseleit buffer. Cycloheximide was infused in half of the sham and acute hemorrhage SV + SI preparations at 50 micrograms/ml. Venous effluent from all groups were analyzed for prostanoid release by radioimmunoassay. The SV + SI released 10-fold more 6-keto-prostaglandin (PG) F1 alpha than PGE2 and thromboxane. Acute hemorrhage increased SV + SI release of 6-keto-PGF1 alpha 3-fold compared to sham. Cycloheximide abolished the increased 6-keto-PGF1 alpha following acute hemorrhage but not the basal release in the sham group. Indomethacin decreased PG synthesis in all groups by 90%. Sham PG release was dependent on a stable pool of cyclooxygenase with a long half-life and was not affected by cycloheximide treatment. Acute hemorrhage stimulated a rapid induction of enzymes (cyclooxygenase, prostacyclin synthase) responsible for prostacyclin synthesis and release which were dependent on de novo protein synthesis.     

Immediate allergic reactions to amoxicillin

A large group of patients with suspected allergic reactions to beta-lactam antibiotics was evaluated. A detailed clinical history, together with skin tests, RAST (radioallergosorbent test), and controlled challenge tests, was used to establish whether patients allergic to beta-lactam antibiotics had selective immediate allergic responses to amoxicillin (AX) or were cross-reacting with other penicillin derivatives. Skin tests were performed with benzylpenicilloyl-poly-L-lysine (BPO-PLL), benzylpenicilloate, benzylpenicillin (PG), ampicillin (AMP), and AX. RAST for BPO-PLL and AX-PLL was done. When both skin test and RAST for BPO were negative, single-blind, placebo-controlled challenge tests were done to ensure tolerance of PG or sensitivity to AX. A total of 177 patients were diagnosed as allergic to beta-lactam antibiotics. We selected the 54 (30.5%) cases of immediate AX allergy with good tolerance of PG. Anaphylaxis was seen in 37 patients (69%), the other 17 (31%) having urticaria and/or angioedema. All the patients were skin test negative to BPO; 49 of 51 (96%) were also negative to MDM, and 44 of 46 (96%) to PG. Skin tests with AX were positive in 34 (63%) patients. RAST was positive for AX in 22 patients (41%) and to BPO in just 5 (9%). None of the sera with negative RAST for AX were positive to BPO. Challenge tests with AX were performed in 23 subjects (43%) to establish the diagnosis of immediate allergic reaction to AX, and in 15 cases (28%) both skin test and RAST for AX were negative. PG was well tolerated by all 54 patients. We describe the largest group of AX-allergic patients who have tolerated PG reported so far. Diagnosis of these patients can be achieved only if specific AX-related reagents are employed. Further studies are necessary to determine the exact extent of this problem and to improve the efficacy of diagnostic methods.     

A YAC-based transcript map of human chromosome 9q22.1-q22.3 encompassing the loci for hereditary sensory neuropathy type I and multiple self-healing squamous epithelioma

The release of lipoteichoic acid (LTA) and teichoic acid (TA) from a Streptococcus pneumoniae type 3 strain during exposure to ceftriaxone, meropenem, rifampin, rifabutin, quinupristin-dalfopristin, and trovafloxacin in tryptic soy broth was monitored by a newly developed enzyme-linked immunosorbent assay. At a concentration of 10 microg/ml, a rapid and intense release of LTA and TA occurred during exposure to ceftriaxone (3,248+/-1,688 ng/ml at 3 h and 3,827+/-2,133 ng/ml at 12 h) and meropenem (2,464+/-1,081 ng/ml at 3 h and 2,900+/-1,364 ng/ml at 12 h). Three hours after exposure to rifampin, rifabutin, quinupristin-dalfopristin, and trovafloxacin, mean LTA and TA concentrations of less than 460 ng/ml were observed (for each group, P < 0.01 versus the concentrations after exposure to ceftriaxone). After 12 h of treatment, the LTA and TA concentrations were 463+/-126 ng/ml after exposure to rifampin, 669+/-303 ng/ml after exposure to rifabutin, and 1,236+/-772 ng/ml after exposure to quinupristin-dalfopristin (for each group, P < 0.05 versus the concentrations after exposure to ceftriaxone) and 1,745+/-1,185 ng/ml after exposure to trovafloxacin (P = 0.12 versus the concentration after exposure to ceftriaxone). At 10 microg/ml, bactericidal antibacterial agents that do not primarily affect cell wall synthesis reduced the amount of LTA and TA released during their cidal action against S. pneumoniae in comparison with the amount released after exposure to beta-lactams. Larger quantities of LTA and TA were released after treatment with low concentrations (1x the MIC and 1x the minimum bactericidal concentration) than after no treatment for all antibacterial agents except the rifamycins. This does not support the concept of using a low first antibiotic dose to prevent the release of proinflammatory cell wall components.     

Bacterial steroidogenesis by periodontal pathogens and the effect of bacterial enzymes on steroid conversions by human gingival fibroblasts in culture

Studies were performed to investigate the effect of microbial culture supernatants of periodontal pathogens on the metabolism of radiolabelled testosterone in the presence or absence of human gingival fibroblasts. Subgingival plaque samples were obtained on paper points from 3 sites with probing depth values of 6-8 mm. Samples were incubated with 14C-testosterone for 24 h in brain heart infusion (BHI) broth. Similar incubations were also carried out with strains of A. actinomycetemcomitans, P. Intermedius and P. gingivalis to study the metabolism of radiolabelled testosterone by these periodontal pathogens. At the end of a 24 h incubation period with fibroblasts and supernatants or sonicates, the radioactive metabolites were extracted with ethyl acetate, evaporated and subjected to thin layer chromatography. The separated metabolites were quantified by scanning the radioactive plates using a Berthold linear analyser. When three sub-gingival plaque samples were incubated with radiolabelled testosterone there were 50-fold, 10-12-fold and 15-17-fold increases in 5 alpha-dihydrotestosterone (DHT) synthesis over 4-androstenedione production in these mixed microbial cultures. The two strains of P. intermedius produced 3- and 20-fold increases in 4-androstenedione production and DHT synthesis respectively. Both strains of A. actinomycetemcomitans and P. gingivalis showed 3-4-fold and 12-28-fold increases respectively in 4-androstenedione synthesis over that of DHT. Culture supernatants of P. intermedius and P. gingivalis caused 3-fold and 2-fold increases in DHT synthesis by fibroblasts over controls. There was little change in the case of the third pathogen. Since DHT has implications on matrix synthesis by fibroblasts in the environment of plaque associated inflammatory periodontal disease, bacterial metabolism and the effect of bacterial supernatants on human gingival fibroblasts can influence the degree of inflammatory repair.     

Synergy and resistance to synergy between beta-lactam antibiotics and glycopeptides against glycopeptide-resistant strains of Enterococcus faecium

A synergistic effect between vancomycin or teicoplanin and different beta-lactam antibiotics was found for two strains of Enterococcus faecium, EFM4 and EFM11, expressing resistance to glycopeptides and belonging to the VANA class. The MICs of penicillin for these two strains were 16 and 128 micrograms/ml, respectively. By using a penicillin-binding protein (PBP) competition assay, it was shown that the affinities of PBPs for different beta-lactam antibiotics and the MICs of these antibiotics obtained in the presence of teicoplanin correlated with the substitution of two high-molecular-weight PBPs for the low-molecular-weight PBP5 as the essential target. Mutants of EFM4 and EFM11 which had lost the synergistic effect between beta-lactams and glycopeptides were selected on teicoplanin plus ceftriaxone at a frequency of 10(-5) and 10(-3), respectively. The mechanism of the loss of synergy was explored. For the mutants derived from EFM4, it was associated with a change in PBPs, while for the mutants derived from EFM11, it was related to some unknown change on the conjugative plasmid responsible for the glycopeptide resistance. These combined observations reflect the relationship which seems to exist between the new D-lactate peptidoglycan precursor, synthesized when the vancomycin resistance is expressed, and the affinity of the different PBPs for this precursor.     

Endothelial cell activation by leukocyte microparticles

The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 +/- 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 +/- 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-alpha, IL-1beta, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or beta2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.     

Condom effectiveness

In this study the effects of indomethacin on secondary inflammatory reaction and secretory function of synoviocytes were analyzed. The results show that secondary inflammatory reaction in adjuvant arthritis rats on days 16, 20 and 22 was suppressed significantly by intragastric administration of indomethacin in a dose of 2 mg/kg-1/d-1 for 10 days. Synoviocytes from adjuvant arthritis rats released a higher level of interleukin (IL)-1 and tumor necrosis factor (TNF) than that from the normal, control group. The synoviocyte culture supernatants of adjuvant arthritis rats were able to enhance the proliferation of synovial fibroblasts from normal rats in vitro. The synovial fibroblast proliferation mediated by the synoviocyte culture supernatants of adjuvant arthritis rats treated with indomethacin was promoted further because of the inhibition of prostaglandin (PG)E2 synthesis and the enhancement of IL-1 and TNF production in the synoviocytes from these animals. These results suggest that indomethacin is an effective antiinflammatory agent, but it is disadvantageous to the repair of joint destruction.     

Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells

This study investigated the signal transduction mechanisms of angiotensin-(1-7) [Ang-(1-7)]- and Ang II-stimulated arachidonic acid (AA) release for prostaglandin (PG) production in rabbit aortic vascular smooth muscle cells. Ang II and Ang-(1-7) enhanced AA release in cells prelabeled with [3H]AA. However, 6-keto-PGF1 alpha synthesis produced by Ang II was much less than that caused by Ang-(1-7). In the presence of the lipoxygenase inhibitor baicalein, Ang II enhanced production of 6-keto-PGF1 alpha to a greater degree than Ang-(1-7). Angiotensin type (AT)1 receptor antagonist DUP-753 inhibited only Ang II-induced [3H]AA release, whereas the AT2 receptor antagonist PD-123319 inhibited both Ang II- and Ang-(1-7)-induced [3H]AA release. Ang-(1-7), receptor antagonist D-Ala7-Ang-(1-7) inhibited the effect of Ang-(1-7), but not of Ang II. In cells transiently transfected with cytosolic phospholipase A2 (cPLA2), mitogen-activated protein (MAP) kinase or Ca(++)-/cal-modulin-dependent protein (CAM) kinase II antisense oligonucleotides, Ang-(1-7)- and Ang II-induced [3H]AA release was attenuated. The CaM kinase II inhibitor KN-93 and the MAP kinase kinase inhibitor PD-98059 attenuated both Ang-(1-7)- and Ang II-induced cPLA2 activity and [3H]AA release. Ang-(1-7) and Ang II also increased CaM kinase II and MAP kinase activities. Although KN-93 attenuated MAP kinase activity, PD-98059 did not affect CaM kinase II activity. Both Ang II and Ang-(1-7) caused translocation of cytosolic PLA2 to the nuclear envelope. These data show that Ang-(1-7) and Ang II stimulate AA release and prostacyclin synthesis via activation of distinct types of AT receptors. Both peptides appear to stimulate CaM kinase II, which in turn, via MAP kinase activation, enhances cPLA2 activity and release of AA for PG synthesis.     

Nitric oxide in the contractile action of bradykinin, oxytocin, and prostaglandin F2 alpha in the estrogenized rat uterus

Experiments were performed on uteri from estrogen-primed female rats. Bradykinin (BK) (10(-8) M) significantly augmented biosynthesis of prostaglandin F2 alpha (PGF2alpha) and prostaglandin E2 (PGE2), and this synthesis was completely blocked by NG-monomethyl L-arginine (NMMA) (300 microM), a competitive inhibitor of nitric oxide synthase (NOS). Blockade of prostaglandin synthesis by indomethacin caused rapid dissipation of isometric developed tension (IDT) induced by BK. Blockade of NOS with NMMA had similar but less marked effects. Combining the two inhibitors produced an even more rapid decay in IDT, suggesting that BK-induced NO release maintains IDT by release of prostanoids. The decline of frequency of contraction (FC) was not significantly altered by either indomethacin or NMMA but was markedly accelerated by combination of the inhibitors, which suggests that PGs maintain FC and therefore FC decline is accelerated only when PG production is blocked completely by combination of the two inhibitors of PG synthesis. The increase in IDT induced by oxytocin was unaltered by indomethacin, NMMA or their combination indicating that neither NO nor PGs are involved in the contractions induced by oxytocin. However, the decline in FC with time was significantly reduced by the inhibitor of NOS, NMMA, suggesting that FC decay following oxytocin is caused by NO released by the contractile process. In the case of PGF2alpha, NMMA resulted in increased initial IDT and FC. The decline in FC was rapid and dramatically inhibited by NMMA. Receptor-mediated contraction by BK, oxytocin, and PGF2alpha is modulated by NO that maintains IDT by releasing PGs but reduces IDT and FC via cyclic GMP.     

Biosynthesis of sulfidopeptide leukotrienes via the transfer of leukotriene A4 from polymorphonuclear cells to bovine retinal pericytes

Administration of exogenous sulfidopeptide leukotrienes (LTs) is associated with enhanced microvascular permeability. In addition, endogenous LTs have been implicated as participants in permeability (nonhydrostatic) edema formation. The source of LTs for interaction with the microvasculature is, however, unknown. We hypothesized that pericytes contribute to vascular LT synthesis. Under basal conditions and after incubation with either the calcium ionophore, A23187 (0-1 microM), or arachidonic acid (20 microM), bovine retinal pericytes (BRPs) did not produce significant amounts of sulfidopeptide LTs. In contrast, in the presence of polymorphonuclear leukocytes (PMNs), which can synthesize LTA4, but not sulfidopeptide leukotrienes, incubation of BRPs with A23187 resulted in dose-dependent increases in LTC4/D4/E4 production (peak: 35.4 +/- 5 pg/microg protein; n = 12). Similarly, BRPs, incubated with exogenous, authentic LTA4 (10 microM), synthesized sulfidopeptide LTs (peak: 18.9 +/- 5 pg/microg protein, n = 3). Preincubation (30 min) of BRPs with PMNs and the lipoxygenase inhibitor, esculetin (1 x 10(-)4 M; n = 12), reduced peak A23187-induced production of LTs by 63.9 +/- 7%. Finally, Northern blot analysis revealed mRNA for 5-lipoxygenase to be present in human and bovine PMNs, but not in BRPs. These results suggest that pericytes produce sulfidopeptide LTs only when provided with LTA4 from an external source such as the PMN. Interactions between pericytes and PMNs may lead to the production of sulfidopeptide LTs, which, in turn, could alter microvascular permeability.     

Histamine and eicosanoid levels in plasma and peripheral blood leucocyte cultures during experimental infection of cattle with bluetongue virus serotype 11

Three calves were sensitized with three doses of inactivated BTV-11 UC8 strain and then experimentally infected with the homologous virus. In addition, four BTV-seronegative heifers were also experimentally infected with BTV-11. Granulocyte rich fractions of peripheral blood leucocyte (PBL-GRF) cultures from BTV-sensitized/infected calves and from control unexposed cattle were exposed in vitro with BTV-11. Histamine, leukotriene (LT) C4 and prostaglandin (PG) D2 were assayed in supernatant fluids. Plasma histamine levels increased in BTV-infected heifers from 10.1 +/- 2 ng/ml at Day 0 to 23.1 +/- 6.6 ng/ml at Day 12 following virus exposure. In addition, in this experimental group the concentration of PGF2 alpha (mean 551.97 +/- 243.54 pg/ml) increased significantly (P < or = 0.05) compared with control cattle (mean 467.3 +/- 73.9 pg/ml). Bluetongue virus induced histamine and LTC4 release after in vitro infection of PBL-GRF. Release of LTC4 was significantly (P < or = 0.05) higher in PBL-GRF cultures from sensitized and control animals than in unexposed PBL-GRF cultures. In contrast to these results, PGD2 was not released after BTV infection of PBL-GRF in vitro. The histamine release caused by BTV was virus-specific and mainly mediated by an immunological reaction, since the release was significantly reduced by removal of cell surface immunoglobulins.     

Induction of nitric oxide production by polyosides from the cell walls of Streptococcus mutans OMZ 175, a gram-positive bacterium, in the rat aorta

The cardiovascular dysfunctions associated with septic shock induced by gram-negative or gram-positive bacteria (gram-positive or gram-negative septic shock) are comparable. In gram-negative septic shock, lipopolysaccharide (LPS) induces nitric oxide (NO) synthase, which contributes to the vascular hypotension and hyporeactivity to vasoconstrictors. The role of NO in gram-positive septic shock and the nature of the bacterial wall components responsible for the vascular effects of gram-positive bacteria are not well known. This study investigated the vascular effects of cell wall serotype polyosides, rhamnose glucose polymers (RGPs), from Streptococcus mutans, in comparison with lipoteichoic acid (LTA) from Staphylococcus aureus, on the induction of NO synthase activity in the rat aorta. We show that 10 microg of both RGPs and LTA per ml induced hyporeactivity to noradrenaline, L-arginine-induced relaxation, increases of 2.2- and 7.8-fold, respectively, of cyclic GMP production, and increases of 7- and 12-fold in nitrite release. All of these effects appeared after several hours of incubation and were inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Electron paramagnetic resonance spin trapping experiments demonstrated directly that RGPs and LTA induced NO overproduction (four- to eightfold, respectively) in rat aortic rings; this production was inhibited by L-NAME and prevented by dexamethasone. These results demonstrate directly the induction of NO production in vascular tissue by LTA and show that another, chemically different component of gram-positive bacteria can also have these properties. This result suggests that different components of the gram-positive bacterial wall could be implicated in the genesis of cardiovascular dysfunctions observed in gram-positive septic shock.     

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid

Osteogenic protein-1 (OP-1), a member of the TGF-beta family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused approximately 2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; approximately 40% vs. approximately 20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by approximately 2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo.     

Nitric oxide: description of a pipeline delivery system

The effect of azelastine hydrochloride (azelastine) on synthesis and release of platelet activating factor (PAF) in alveolar macrophages obtained from asthmatic and non-asthmatic subjects was examined. Alveolar macrophages (AMs) were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 microM) for 15 min. PAF activity was detected by aggregation of washed guinea pig platelets. PAF activity released from alveolar macrophages (AMs) from asthmatics without preincubation of azelastine was 15.97 [2.17] (mean [SD], ng/10(7) cells) in supernatants and 42.52 [10.16] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF activity reduced to 10.71 [2.73] (mean [SD], ng/10(7) cells), 7.86 [0.94], and 3.52 [0.31] in the supernatants, and 35.58 [7.37], 21.57 [4.36], and 14.77 [0.99] (n = 15) in the cell pellets, respectively. PAF activity in non-asthmatic subjects without preincubation of azelastine was 8.55 [1.16] (mean [SD], ng/10(7) cells) in supernatants and 32.64 [3.37] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF activity reduced to 6.68 [0.78] (mean [SD], ng/10(7) cells), 4.47 [0.51], and 2.97 [0.36] in the supernatants, and 29.53 [3.75], 14.78 [1.95], and 6.16 [0.55] (n = 20) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra- and extracellular PAF activity from asthmatic and non-asthmatic macrophages in the same manner.     

beta-Lactam synthetase: a new biosynthetic enzyme

The principal cause of bacterial resistance to penicillin and other beta-lactam antibiotics is the acquisition of plasmid-encoded beta-lactamases, enzymes that catalyze hydrolysis of the beta-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of beta-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical beta-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg2+-dependent enzyme, a beta-lactam synthetase (beta-LS). Insertional inactivation of its encoding gene in wild-type Streptomyces clavuligerus resulted in complete loss of clavulanic acid production and the accumulation of N2-(carboxyethyl)-L-arginine (CEA). Chemical complementation of this blocked mutant with authentic deoxyguanidinoproclavaminic acid (DGPC), the expected product of the beta-LS, restored clavulanic acid synthesis. Finally, overexpression of this gene gave the beta-LS, which was shown to mediate the conversion of CEA to DGPC in the presence of ATP/Mg2+. Primary amino acid sequence comparisons suggest that this mode of beta-lactam formation could be more widely spread in nature and mechanistically related to asparagine synthesis.     

Multivariate analysis of risk factors for infection due to penicillin-resistant and multidrug-resistant Streptococcus pneumoniae: a multicenter study

Pneumococcal disease was studied prospectively to determine the risk factors associated with resistance to penicillin and other antibiotics. One hundred twelve clinically significant pneumococcal isolates were recovered from 95 patients. Approximately one-half (49.47%) of the cases were due to penicillin-resistant strains. Multivariate analysis showed that previous use of beta-lactam antibiotics (odds ratio [OR], 2.81; 95% confidence interval [CI], 0.95-8.27), alcoholism (OR, 5.22; 95% CI, 1.43-19.01), and noninvasive disease (OR, 4.53; 95% CI, 1.54-13.34) were associated with penicillin resistance, whereas intravenous drug use (OR, 0.14; 95% CI, 0.03-0.74) was not. Statistical analyses of the variables associated with resistance to multiple antibiotics detected age of younger than 5 years (OR, 16.79; 95% CI, 1.60-176.34) or of 65 years or older (OR, 4.33; 95% CI, 1.42-13.21) and previous use of beta-lactam antibiotics by patients with noninvasive disease (OR, 7.92; 95% CI, 1.84-34.06) as parameters associated with increased risk. We conclude that multivariate analysis provides clues for empirical therapy for pneumococcal infection.     

Stimulation of prostaglandin E2 production by interleukin-1 alpha and transforming growth factor alpha in osteoblastic MC3T3-E1 cells

The mechanism by which interleukin-1 (IL-1) and transforming growth factor alpha (TGF-alpha) regulate prostaglandin synthesis has been examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in DMEM containing 10% fetal calf serum. Prostaglandin E2 (PGE2) production was determined by radioimmunoassay or by prelabeling cells with [3H]arachidonic acid, followed by high-performance liquid chromatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by northern blot analysis using [32P]labeled cDNA probes. By HPLC, PGE2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6-keto-PGF1 alpha into the medium was detected. PGE2 production was stimulated approximately 7- to 14-fold by IL-1 (1 ng/ml) and 3- to 8-fold by TGF-alpha (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-alpha caused a synergistic 37- to 71-fold increase in PGE2 accumulation. PGHS-1 mRNA levels were maximally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fold by TGF-alpha after 24 h; the combination of IL-1 and TGF-alpha produced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in response to combined IL-1 and TGF-alpha. PGHS-2 mRNA was increased 1.4-fold by TGF-alpha at 1 h, and the combination of IL-1 and TGF-alpha caused a 1.7-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL

Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of ftsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of beta-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.