Surface action of gentamicin on Pseudomonas aeruginosa

The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial protein synthesis alone appears to be insufficient to entirely explain the bactericidal effects. Bacteriolysis is also mediated through perturbation of the cell surface by gentamicin (J.L. Kadurugamuwa, J.S. Lam, and T.J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993). In order to separate the surface effect from protein synthesis in Pseudomonas aeruginosa PAO1, we chemically conjugated bovine serum albumin (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytoplasm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study the surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibiotic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro but not on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold particles were clearly seen on the cell surfaces of whole mounts and thin sections of cells, while the cytoplasm was devoid of such particles. Disruption of the cell envelope was also observed since gentamicin-BSA and gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antibiotic disrupts the packing order of lipopolysaccharide of the outer membrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially lethal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from surface perturbation; the two effects in concert make aminoglycoside drugs particularly effective antibiotics.     

Direct vasodilatory effect of insulin on isolated retinal arterioles

PURPOSE: To test the hypothesis that insulin has a direct vasodilatory effect on retinal arteries and their branches and to investigate the mechanisms involved. METHODS: Segments of porcine retinal arteries were dissected, cannulated, and perfused. Vessel diameter was measured continuously on-line. Vessels were precontracted to 66% +/- 0.9% (SEM, n = 148) of their original diameter by perfusing with 124 mM K(+)-Krebs solution. Dose-response curves to insulin (2 to 2000 microU/ml) were compared for extraluminal (EL), intraluminal (IL), and combined IL-EL application. The effect of cyclooxygenase and nitric oxide synthase inhibition on the insulin response was determined, as was Ca2+ channel involvement. RESULTS: EL insulin alone had no significant effect on vessel diameter. IL insulin produced a dose-dependent dilatation of 5.6% +/- 2.9% (n = 22) of the K+ contracted diameter at 200 microU/ml and up to 12.4% +/- 3.6% (n = 22) by 2000 microU/ml, whereas combined IL-EL insulin application caused dilatation at all concentrations, rising to 15.1% +/- 2.9% (n = 44) at 200 microU/ml and 19.7% +/- 3% (n = 44) at 2000 microU/ml. IL indomethacin (5 x 10(-5) M) had no significant effect on the insulin-induced dilatation, whereas IL L-NAME (10(-4) M) inhibited insulin dilatation completely. The addition of EL verapamil (10(-6) M) during insulin-induced dilatation resulted in further dilatation to 37.8% +/- 4.2% (n = 18). However, the addition of insulin to verapamil-dilated vessels caused no further dilatation. Exposure to EL insulin while the IL K+ contraction dose-response curve was measured had no effect. Results in main arteries and branches did not differ. CONCLUSIONS: The IL application of insulin dilates potassium-contracted pig retinal arteries. This effect was enhanced by the EL presence of insulin, which did not result in dilatation when it was administered alone. The dilatation response was mediated by nitric oxide but not by prostaglandins. There was some evidence for the involvement of Ca2+ channels in insulin-induced dilatation. These results imply that insulin is a vascular regulator in normal conditions and may have relevance to the vascular changes occurring in diabetes and hypertension in the retina.     

The effect of hydroxyethyl starch on platelet aggregation in vitro

To elucidate the role of endogenous nitric oxide (NO) in allergen- (AIB) and hyperventilation-induced bronchoconstriction (HIB), the effects of an NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on AIB and HIB were studied in guinea-pigs. In the AIB group, 21 anaesthetized guinea-pigs, actively sensitized with 1% ovalbumin, were challenged with aerosolized 0.1% ovalbumin solution under mechanical ventilation. In the HIB group, 14 guinea-pigs were challenged with hyperventilation (tidal volume of 12 mL x kg(-1) at 150 breaths x min(-1) with 21% O2 and 5% CO2 dry gas) for 5 min. In both groups, lung resistance (RL) was measured using a pressure-volume-sensitive body plethysmograph, with or without L-NAME pretreatment (8 mg x kg(-1) i.v. followed by 2 mg x kg(-1) x min(-1) i.v.). The NO precursor, L-arginine was injected at a rate of 15 mg x kg(-1) x min(-1) after L-NAME injection (10 mg x kg(-1)) in the AIB group. The results were as follows. In the AIB group, the maximal RL change was significantly potentiated by pretreatment with L-NAME. This potentiating effect of L-NAME was reversed by L-arginine. In the HIB group, the pretreatment with L-NAME had no effect on increases in RL. These findings suggest that endogenous nitric oxide may play an important role in the modulation of allergen-, but not hyperventilation-induced bronchoconstriction in guinea-pigs.     

The F2-isoprostane 8-epiprostaglandin F2alpha increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.     

Retinoic acid-induced abnormal development of hindlimb joints in the mouse

We asked if the amount of SP-B (range 37-410 micrograms/ml) in surfactants used to treat preterm lambs at 123 days of gestation correlated with postnatal lung function or the SP-B content of surfactant recovered by alveolar washes after 10 h ventilation. Ventilation was initiated using a low tidal volume strategy to minimize early lung injury. There were small increases in compliance for the lambs treated with surfactants containing more than 37 micrograms/ml SP-B and an increased lung volume for lambs treated with a surfactant containing 385 micrograms/ml/ml relative to the surfactant containing 37 micrograms/ml SP-B. The amount of SP-B in the surfactant used for treatment correlated linearly with the amount of SP-B in the surfactant recovered from the lambs (r = 0.95, p < 0.001). Albumin leak from the vasculature to the airspace was low as was total protein in alveolar washes, indicating minimal lung injury. The SP-B content of surfactant (from 37 to 410 micrograms/ml) had minimal effects on postnatal lung function over a 10-hour study period in lambs ventilated in a manner to minimize lung injury.     

The effect of light deprivation on the mouse lens

This work was undertaken to test the hypothesis that first exposure of the eye to light is responsible for the changes in lens protein expression patterns observed around the time of birth. The effect of light deprivation on lens properties was examined in Balb c mice which were bred, reared and maintained in complete darkness for up to 7 months. Data were collected on body weight, lens weight, lens protein contents and crystallin distributions. The data were compared with those obtained from age matched mice maintained in natural light/dark conditions. No significant differences were observed in body weight between animals maintained in the light and dark. However, animals kept in the dark had significantly smaller lenses. After 6 months in the dark, the lens represented 0.02% of body weight compared with 0.031% in the light reared animals (P < 0.001). Lens protein concentration, insoluble protein contents and crystallin synthesis patterns were indistinguishable for the two groups of animals. It is concluded that light stimulation of the eye is required for optimal lens growth but does not affect the production of specific crystallins.     

Beneficial effect of ursodeoxycholic acid on mucosal damage in trinitrobenzene sulphonic acid-induced colitis

BACKGROUND: Recently we observed that ursodeoxycholic acid (UDCA) ameliorates an experimental small intestinal inflammation induced by indomethacin in the rat. In this study, we have tested whether ursodeoxycholic acid also reduces mucosal damage in the bile-independent trinitrobenzene sulphonic acid (TNB) model of experimental colitis. METHODS: Intestinal inflammation (colitis) was induced in male Sprague-Dawley rats (250-300 g) by intracolonic administration of TNB (30 mg in 50% ethanol). Rats were treated with UDCA (10 mg/kg) either for 3 days starting with the administration of TNB for an acute inflammation (n = 11) or for 8 days starting one day after induction of colitis related to a more acute/chronic inflammation (n = 11). Rats were sacrificed at day 3 or day 9, respectively. Healing of induced colitis was assessed by macroscopic and blinded microscopic analysis as well as by measurement of bowel wet weight, daily body weight, and myeloperoxidase activity. All examinations were separately performed in three colon segments (S1 3-5 cm, S2 5.5-8 cm and S3 8.5-11 cm from anus). RESULTS: UDCA treatment significantly reduced macroscopically and microscopically detectable injury in acute inflammation in segments 1 and 2. The colitis-rats with acute/chronic inflammation had less marked mucosal damage. Nevertheless, UDCA treatment led to a significant decrease of visible injury parameters which was seen exclusively at the area of maximal ulceration (S2). Furthermore, a significant increase in body weight of UDCA-treated TNB rats compared to controls from day 5 on was found. CONCLUSION: Ursodeoxycholic acid attenuates the severity of acute inflammation and inhibits the development of acute/chronic inflammation predominantly around the area of maximal ulceration in TNB-induced colitis. In addition to our previous studies and results in indomethacin induced enteritis, these data may provide a rationale for studying how UDCA modulates functions of immune cells in the colonic mucosa.     

Role of nitric oxide in kainic acid-induced elevation of cochlear compound action potential thresholds

PTH-related protein (PTHrP) is found with its receptor in a variety of normal mammalian embryonic tissues where it apparently regulates cellular growth and differentiation. PTHrP stimulates phosphatidylcholine synthesis in rat fetal lung explants, suggesting a role in fetal type II alveolar maturation and surfactant production. We investigated PTHrP levels in tracheal aspirates of newborn infants. We collected tracheal aspirates from 40 intubated newborn infants within the first 24 h of life. PTHrP levels were measured by a RIA using rabbit antisera to PTHrP peptide 38-64. We found significantly lower PTHrP levels in tracheal aspirates from infants born at less than 35 wk of gestation (p = 0.02) and with a birth weight less than 2 kg (p = 0.04). We also found significantly lower PTHrP levels in male preterm (<35 wk of gestation) infants compared with female infants (p = 0.01), and in preterm infants who required multiple doses of surfactant (p = 0.005). Preterm infants exposed to antenatal steroids had significantly higher levels of PTHrP in tracheal aspirates (p = 0.02). PTHrP is associated with various indices of lung maturation and may prove to be a mediator of differentiation and growth.     

Long term effects of 50 mg acetylsalicylic acid alone and in combination with dipyridamole on platelet function after coronary bypass surgery

In a prospective randomized trial in 42 patients undergoing coronary artery bypass surgery, we analyzed the long term platelet inhibiting effects of 50 mg acetylsalicylic acid (ASA) by itself and in combination with dipyridamole (2 x 200 mg), in comparison with phenprocoumon. Three and six months therapy led to significant inhibition of maximum aggregation induced by collagen 1 microgram/ml in platelet rich plasma (PRP) by more than 50% (p < or = 0.05). In PRP stimulated with 5 micrograms/ml collagen maximum inhibition amounted to nearly 20% (n.s.). The groups treated with ASA/ASA + dipyridamole showed an ADP threshold concentration 2.5 times higher than the group treated with phenprocoumon (p < or = 0.05). After stimulation with collagen 1 microgram/ml and 5 micrograms/ml thromboxane B2 synthesis in vitro in both groups treated with ASA was reduced to 1% of the base line values (p < or = 0.01). Inhibition of aggregation in whole blood appeared evident, but was not statistically significant due to considerable fluctuation of measurement. An additional effect of dipyridamole was not detectable. In conclusion, treatment with 50 mg ADA/d results in a lasting, effective inhibition of aggregation of platelets in patients with coronary artery bypass surgery. There is no synergistic effect of additional dose of 400 mg dipyridamole/d.     

The effect of gentamycin on immunity in immunodeficiency and the action of immunomodulators

The effect of 3 and 6 mg/kg gentamicin on the immunity of intact mice and cyclophosphan- and azathioprine-induced immunodeficiency was studied. Gentamicin deepens the severe suppression of antibody genesis against the background of cyclophosphan and azathioprine, inhibits the reaction of GZT in intact mice. The antibiotic does not prevent stimulation of AOK production by methyluracil and prodigiosan, and does not change the effect of immunostimulators on GZT in immunosuppression. Gentamicin has no effect on the phagocytic activity of neutrophils and macrophages in intact animals. The character of the effect of the antibiotic on phagocytosis depends on the genesis of immunosuppression. The effectiveness of gentamicin in experimental infection is maintained in immunosuppression also.     

Comparison of activity of sisomicin and gentamicin in mouse protection tests with gram-negative bacilli

The efficacy of sisomicin and gentamicin was compared in mouse protection studies against strains of Escherichia coli, Klebsiella sp., Enterobacter aerogenes, Serratia marcescens, Proteus mirabilis, and Pseudomonas aeruginosa. There was no significant difference in mortality of the mice in any of the protocol groups when five different dosages of sisomicin and gentamicin given by three separate schedules were compared for each bacterial inoculum in each antibiotic protocol. The mean protective dose values of sisomicin were at least one-half those of gentamicin for each protocol against Pseudomonas aeruginosa.     

Permissive effect of nitric oxide in arachidonic acid induced dilation in isolated rat arterioles

OBJECTIVE: Prostaglandins and nitric oxide play an important role in the regulation of arteriolar tone. L-Arginine analogues inhibit nitric oxide formation, but may also inhibit arachidonic-acid induced dilation. Nitric oxide was found to stimulate cyclooxygenase activity in cultured endothelial cells. Therefore, we hypothesized that the non-specific inhibition of prostaglandin-related dilation by L-arginine analogues is a consequence of the absence of nitric oxide. METHODS: To test this hypothesis, arteriolar segments from rat cremaster muscle were studied in a pressure myograph at 75 mmHg. Segments developed spontaneous tone, the diameter reduced from 179 +/- 3 to 98 +/- 3 microns (n = 41). In this condition, responses to exogenous arachidonic acid (1 microM) were recorded and compared with responses after addition of L-NNA, and addition of either SNAP, nitroprusside or 8-Br-cGMP in the presence of L-NNA. RESULTS: Inhibition of basal nitric oxide production with L-NNA (0.1 mM) reduced arachidonic acid-induced dilation (from 52 +/- 9 to 31 +/- 6 microns). In the presence of L-NNA, responses to arachidonic acid were augmented when exogenous nitric oxide was also present (SNAP, 31 +/- 6 microns vs. 75 +/- 5 microns; nitroprusside, 31 +/- 8 microns vs. 42 +/- 7 microns). Responses were not augmented with the second messenger of nitric oxide-mediated dilation 8-Br-cGMP (37 +/- 9 microns vs. 32 +/- 9 microns). CONCLUSIONS: These results indicate that nitric oxide directly increases arachidonic acid-induced dilation. Thus, the non-specific effect of L-arginine analogues can be explained by a permissive effect of nitric oxide on endothelial arachidonic acid metabolism.     

The strain effect of dam on intrauterine incisal growth in mouse fetuses

The embryo transfer technique was carried out to examine the strain effect of dams on the intrauterine incisal growth of mouse fetuses. A mean of nine DDD/Qdj embryos was transferred to each female recipient of the four strains (DDD/Qdj, C3H/Qdjl C57BL/Qdj, and DBA/IJ Sea). The dam's weight, the litter size, the gestation period and the newborn offspring weight were recorded. The mandibular incisal size of the newborn offspring was measured two-dimensionally by means of an image analyzing system. Statistical analyses of the incisal size of the DDD/Qdj newborn offspring delivered from the four strains of recipients showed the following results: 1) There were significant interstrain differences in the dam's weight and the newborn offspring weight, but not in the litter size and the gestation period among the four strains of recipients. 2) There was an inverse relation between the litter size and the mandibular incisal size while there were direct relations between the dam's weight and the mandibular incisal size, and between the newborn offspring weight and the mandibular incisal size. 3) There was a significant strain effect of the dam on the mandibular incisal size of the newborn offspring. 4) After eliminating the effects of the litter size, the gestation period, and the newborn offspring weight on the mandibular incisal size of the DDD/Qdj newborn offspring developed in the four strains of dams, a significant interstrain difference in the adjusted mandibular incisal size was found (C3H/Qdj > DBA/IJ Sea > C57BL/Qdj > DDD/Qdj), suggesting that the uteri of the C3H/Qdj strain of dams provided a more suitable environment for the intrauterine incisal growth than those of the other three strains of dams. Thus, it is concluded that the strain effect of dam played an important role in the intrauterine incisal growth of mouse fetuses.     

The effect of ulinastatin on CFU-C colony formation in mouse myelocyte cultures

The mixture of 5'-guanylylimidodiphosphate (Gpp(NH)p)/EDTA/NaCl has been used to delineate low-affinity conditions for agonists binding to G-protein-linked receptors. The effects of this mixture on [3H]RX821002 (2-methoxyidazoxan) binding to alpha2-adrenoceptors were evaluated in different tissues. The density of alpha2-adrenoceptors in the presence of the mixture was 11, 78 and 60% higher in human cortex (predominant alpha2A), human caudate (alpha2A + alpha2C) and rat kidney (alpha2A + alpha2B), respectively, than in its absence. In rat kidney, masking of alpha2B-adrenoceptors by ARC239 (2-[2-[4-(o-methoxyphenyl)-piperazin-1-yl]-ethyl]-4,4-dimethyl-1,3 -(2H,4H)-isoquinolindione) (50 nM) or masking of alpha2A-adrenoceptors by BRL44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidaz ole) (100 nM) demonstrated that the increase was in the alpha2B-adrenoceptor but not in the alpha2A-adrenoceptor subtype.     

The mode of action of homocysteine on mouse brain glutamic decarboxylase and gamma-aminobutyrate aminotransferase

In the belief that homocysteine-induced convulsions might be related to alterations in brain gamma-aminobutyric acid metabolism, we have studied the action of this amino acid on the activity of glutamic decarboxylase (GAD, EC 4.1.1.15) and gamma-aminobutyrate aminotransferase (EC 2.6.1.19) of mouse brain in vitro DL-homocysteine competitively inhibited GAD with respect to both L-glutamate and pyridoxal 5'-phosphate. The respective Ki's were 3.8 mM and 0.3 mM. The activity of GABA-T also was altered in the presence of DL-homocysteine. A competitive inhibition (Ki = 6 mM) was observed with gamma-aminobutyric acid, and an uncompetitive inhibition with respect to pyridoxal 5'-phosphate and alpha-ketoglutarate. These results are explained in terms of a dual action of homocysteine on each of the enzymes: one involving a competition for substrate binding site and the other involving the formation of an inactive inhibitor-cofactor complex. The significance of the inhibition of these enzymes of gamma-aminobutyric acid metabolism is discussed in relation to the convulsant action of homocysteine.     

Effect of platelet activating factor on embryonic development and implantation in the mouse

Platelet activating factor (PAF) was administered to female mice in order to investigate its effect on ovulation rate and on oocyte quality including their in-vitro embryonic development, implantation and uterine receptivity. In experiment 1, 4-week-old female mice were assigned to receive PAF or phosphate buffered saline for 4 consecutive days. On the second day of this treatment, pregnant mares' serum gonadotrophin was administered and human chorionic gonadotrophin (HCG) 48 h later, after which copulation occurred. Oocytes were collected on the following day and evaluated. The mean number of oocytes and zygotes (two pronuclear stage embryos) recovered from the PAF-treated group was not different from the control group (31 versus 27), but the proportion of zygotes was higher in PAF-treated group than in controls (83 versus 68%, P < 0.05, PAF versus controls). Although the rate of in-vitro first cleavage was not different in the two groups (82 versus 69% respectively), hatching was higher in the PAF-treated group than control mice (99 versus 83%, P < 0.01). In experiment 2, the in-vitro developed blastocysts from experiment 1 were transferred into the uterus of day 3 pseudopregnant PAF-treated or control recipients. Three different combinations of intrauterine transfer were performed; PAF embryo to control recipient (PAF-->C: n = 19), control embryo to PAF recipient (C-->PAF: n = 19), and control embryo to control recipient (C-->C: n = 22). Implantation and abortion were assessed on day 19 posttransfer. The implantation rate of C-->PAF (23.7%) was lower than C-->C (31.1%, P < 0.05), but was not different from PAF-->C (31.2%). Further, C-->PAF showed a higher abortion rate per embryo (29.6%) than PAF-->C (12.7%, P < 0.05), but was not different from C-->C (24.4%). In the present study, PAF administration enables females to produce oocytes with a higher potential for fertilization, in-vitro development and implantation, but has a detrimental effect on uterine receptivity to embryos.