Inhibitory effects of water deficit on maize leaf elongation

The growth rate of maize leaves has been investigated for plants grown in pots in controlled conditions and subjected to slow drying over a period of days. The elongation of leaves experiencing water deficit was inhibited primaily during the afternoon. No decrease in the turgor of the growing cells could be detected at that time. Solute concentration in the growing cells increased in tissue experiencing water deficit, but this was shown to occur after the growth rate had fallen. Calculations of the rate of solute accumulation necessary to maintain these concentrations indicated that the rate was less in slowly growing than in rapidly growing cells. The growing tissue of well-watered leaves excreted protons into the apoplastic space, but this acidification decreased in tissue exposed to water deficit. The pH of the apoplastic space correlated with the growth rate of the tissue. In vitro acidification of isolated, frozen-thawed tissue, maintained under constant tension, increased wall extensibility. The results suggest that one role of proton excretion may be to promote wall-loosening events necessary for cell enlargement, and that inhibition of this process may have reduced growth rate in leaves exposed to water deficit.     

Liposome-enabled synergistic interaction of antimicrobial agents

Antimicrobial agents may interact synergistically when both drugs are present at the infected site for an adequate period of time at sufficient concentrations. Generally speaking, the agents in the combination show different tissue distributions and pharmacokinetics. By co-encapsulation of the drugs in a drug carrier, like liposomes, parallel tissue distributions of both drugs may be ensured and drug concentrations at the site of infection may be increased. In this presentation therapeutic efficacy of liposome-co-encapsulated gentamicin (GN) and ceftazidime (CZ) will be shown in a GN-CZ-susceptible and GN-CZ-resistant Klebsiella pneumoniae-pneumonia in rats.     

LIPOSOME-ENABLED SYNERGISTIC INTERACTION OF ANTIMICROBIAL AGENTS

ABSTRACT

Antimicrobial agents may interact synergistically when both drugs are present at the infected site for an adequate period of time at sufficient concentrations. Generally speaking, the agents in the combination show different tissue distributions and pharmacokinetics. By co-encapsulation of the drugs in a drug carrier, like liposomes, parallel tissue distributions of both drugs may be ensured and drug concentrations at the site of infection may be increased. In this presentation therapeutic efficacy of liposome-co-encapsulated gentamicin (GN) and ceftazidime (CZ) will be shown in a GN-CZ-susceptible and GN-CZ-resistant Klebsiella pneumoniae-pneumonia in rats.     

Differences in amniotic fluid and maternal serum cytokine levels in early midtrimester women without evidence of infection

The amniotic fluid cytokine profile has been shown to be indicative of various disease states, and changes may be associated with preterm labor or infection. Anti-inflammatory cytokine profiles may be essential for successful normal pregnancy. However, there are currently few normative data on the concentration of cytokines in amniotic fluids during pregnancy. The aim of this study was to provide new amniotic fluid cytokine data for future comparative studies in disease states, notably in utero viral infections, and to compare these with maternal serum levels. Amniotic fluid was obtained from 100 pregnant women undergoing elective amniocentesis at the Royal Hospital for Women, Randwick. Concentrations of 27 cytokines were simultaneously measured in amniotic fluid and a subset of matching maternal sera (n=33) using a multiplex bead-based immunoassay system (Bio-Plex, Bio-Rad). To exclude infection, nested multiplex PCR targeting 17 known congenital infectious agents were performed on all amniotic fluid and maternal serum samples, and serological testing was also performed against some of these agents. Maternal serum concentration was positively correlated with amniotic fluid levels for MIP-1beta (r=0.39, P=0.027). IL-1ra was positively correlated to maternal age (r=0.210, P=0.036), and mean IL-5 levels were significantly higher in amniotic fluids from pregnancies with male fetuses than those with female fetuses (P=0.036). Normal amniotic fluid concentrations for five cytokines (IL-6, IL-8, IP-10, MCP-1, IL-1ra) were found to be significantly elevated over maternal serum concentrations in matched pairs (P<0.05). Concentrations of 12 cytokines (eotaxin, IFN-gamma, IL-9, IL-12, IL-15, IL-17, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, VEGF, PDGF bb) were significantly elevated in maternal serum compared to paired amniotic fluid at midtrimester (P<0.05). Amniotic fluid may be more representative of the fetal cytokine profile than cytokine analysis on antenatal sera as it represents predominantly fetal urinary and respiratory secretions. This study provides new normative data for multiple cytokine levels in amniotic fluid and maternal sera at 14-16 weeks gestation, and is a valuable tool for future diagnostic and comparative studies.     

Compromised antioxidant status and persistent oxidative stress in lung transplant recipients.

Oxidative stress may be a key feature, and hence important determinant, of tissue injury and allograft rejection in lung transplant recipients. To investigate this, we determined the antioxidant status (urate, ascorbate, thiols and alpha-tocopherol) and lipid peroxidation status (malondialdehyde) in bronchoalveolar lavage (BAL) fluid and blood serum of 19 consecutive lung transplant recipients 2 weeks and 1, 2, 3, 6, and 12 months post-surgery. BAL fluid and blood samples from 23 control subjects and blood from 8 patients two days before transplantation were obtained for comparison. Before surgery, the antioxidant status of patients was poor as serum ascorbate and total thiol concentrations were significantly (p < 0.05) lower than control subjects. Two weeks post-surgery, ascorbate and total thiol concentrations were still low and urate concentrations had fallen compared to control subjects (p < 0.01). At this time, BAL fluid urate concentration was higher (p < 0.01), ascorbate concentration was lower (p < 0.01) and reduced glutathione concentrations were similar to control subjects. MDA, a product of lipid peroxidation, was higher (p < 0.01) in both BAL fluid and serum obtained from transplant patients compared to control subjects. During the first 12 months post-surgery, little improvement in antioxidant status or extent of lipid peroxidation was seen in transplant recipients. Regression analysis indicated no difference in serum or BAL fluid antioxidant status in patients with acute rejection compared to those without. In conclusion, lung transplant recipients have a compromised antioxidant status before surgery and it remains poor for at least the first year following the operation. In addition, these patients have elevated MDA concentrations in both their lung lining fluid and blood over most of this time. Oxidative stress is not, however, a sufficiently sensitive endpoint to predict tissue rejection in this group.     

Cross sectional survey of effectiveness of lipid lowering drugs in reducing serum cholesterol concentration in patients in 17 general practices

ObjectiveTo compare the effectiveness of lipid lowering drugs in lowering serum cholesterol concentrations.DesignCross sectional study.Setting17 practices within 17 primary care groups in Trent region, United Kingdom.ParticipantsPatients aged 35 years or over taking lipid lowering drugs and with at least two serum cholesterol concentrations recorded on computer.Results1353 of 2469 (54.8%) patients receiving lipid lowering treatment had a last recorded serum cholesterol concentration of ⩽5 mmol/l. Significantly more patients taking statins achieved the target value for serum cholesterol (5 mmol/l) than those taking fibrates (1307 (57%) v 46 (26%); P<0.0001). Atorvastatin and simvastatin were the most effective drugs in achieving the target. Significant differences were found between lipid lowering drugs for the pretreatment serum cholesterol concentration, the most recent cholesterol concentration, and the associated percentage reduction. Atorvastatin and simvastatin achieved the greatest percentage reduction in serum cholesterol concentrations (30.1%, 95% confidence interval 28.8% to 31.4%, and 28.0%, 26.7% to 29.3%, respectively). Although the mean serum cholesterol concentrations in this unselected population tended to be higher than those in clinical trials, the percentage reduction was consistent with the trials.ConclusionThe ability of individual statins to lower serum cholesterol concentration varied, with atorvastatin and simvastatin being the most effective. The percentage reductions agreed with those of randomised controlled trials indicating likely benefits in unselected patients in primary care. As the initial serum cholesterol concentrations were higher than those in randomised controlled trials, target serum cholesterol values of ⩽5 mmol/l may be unrealistic even for patients taking the most efficacious drugs. Also, the higher initial levels could mean that the absolute reduction in cardiovascular risk in primary care patients is greater than thought.

What is already known on this topic

Statins in patients with coronary heart disease help reduce further cardiovascular events and improve survivalThis seems to be a class effect of statins, although there may be important differences in effectiveness between themLess than half of patients in the community who take lipid lowering drugs achieve target serum cholesterol values

What this study adds

Statins vary in their ability to lower serum cholesterol concentration, with atorvastatin and simvastatin achieving the best resultsThe percentage reductions agreed with those found in randomised controlled trialsSince the initial serum cholesterol concentrations were higher than in trials, absolute risk reductions in primary care patients may be greater than thoughtTarget values of ⩽5 mmol/l may be unrealistic even for patients on the most efficacious drugs, because the initial mean cholesterol values of primary care patients are higher than those of patients in trials     

WHOLE-EMBRYO CULTURE AND THE STUDY OF MAMMALIAN EMBRYOS DURING ORGANOGENESIS

1. Simple and reliable methods are now available for growing rat and mouse embryos in culture at all stages of organogenesis. Primitive-streak embryos can be maintained for up to 5 days in culture while they develop to early foetal stages. Older embryos are maintained for progressively shorter periods and the most advanced stage that can be supported is equivalent to the rat foetus of 15 days' gestation. 2. The rates of protein synthesis and differentiation of the younger embryos in vitro are similar, and of head-fold embryos identical, to those in vivo. After the formation of the limb buds growth is slower, with protein synthesis more retarded than differentiation, resulting in embryos or foetuses that are well formed but smaller than in vivo. This slowing of growth of the older embryos in culture is probably caused by the lack of a functional allantoic placenta. 3. The embryos of some other species, including the guinea-pig, hamster, rabbit and opossum have also been maintained in culture during organogenesis but the results are not yet as good as those for rats and mice. 4. Maximum growth of rat embryos explanted with the visceral yolk sac intact is obtained in undiluted homologous serum, though adequate growth for many studies can be maintained in mixtures of serum with chemically defined tissue-culture media. The best results are obtained in serum prepared from blood centrifuged before clotting has occurred (I.C. serum) and heat-inactivated. The importance of a high concentration of serum in the culture medium may be related to the mechanisms for uptake, transport and digestion of macromolecules by the rodent yolk sac. 5. There is no convincing evidence for a changing rate of oxygen consumption during organogenesis but there is strong evidence for changes in energy metabolism. At the beginning of organogenesis, the embryo shows a high rate of anaerobic glycolysis and of pentose-shunt activity. During the following days these decline while activity of the Krebs' cycle and electron-transport system increases. Anoxia, or exposure of the embryo to carbon monoxide, increases glycolysis and reduces growth rate. 6. The earliest stages of the formation of the heart and blood circulation can be closely observed in culture. The heart rate of the 111/2-day rat embryo is about 160 beats per minute at 38°C, and falls by about 7% per degree for lower temperatures. Several drugs that are cardioactive in the adult also affect the frequency of the heartbeat in the embryo, and the pattern of response suggests that the adrenergic receptors in the embryo develop before the cholinergic receptors. Experiments in which embryo and yolk sac were cultured separately, as well as together, have indicated that haemopoiesis can occur in the embryo only after a migration of stem cells from the yolk sac. 7. Microsurgery has been successfully applied to embryos in culture in studies on morphogenetic movements, heart development, axial rotation, limb-bud regeneration and placenta formation. Biochemical studies of normal morphogenesis have been few, but one has shown a high rate of hyaluronate synthesis by the embryo which may be related to the maintenance and expansion of extracellular spaces and the formation of the neural folds. 8. Embryos are particularly sensitive to teratogenic agents during organogenesis. Teratogens that have been studied on whole embryos in culture include trypan blue, antisera, hyperthermia, anaesthetics, and abnormal concentrations of vitamins, oxygen and glucose. Many of the malformations induced have been similar to those obtained after administration of the same agents in vivo and have demonstrated a direct teratogenic effect on the embryo independent of the maternal metabolism. It is suggested that culture methods may provide a valuable additional screening procedure for new drugs and other potentially embryopathic agents.     

Luminal secretion of myo-inositol by the rat epididymis perfused in vitro

A technique for perfusing the lumen of rat epididymal tubules maintained in vitro showed that [3H]inulin was largely excluded from the lumen of unravelled tubules from the cauda and tubules from the corpus if the connective tissue capsule was removed. The preparation transported [3H]inositol from the bath fluid for 3 h against a concentration gradient in both regions with activity rising (16-29% of bath fluid values) in the cauda and reaching a plateau (18%) in the corpus epididymidis. HPLC showed that radioactivity was solely associated with inositol and its movement to the lumen was reduced by raising inositol in the bath fluid from 50 microM (plasma levels) to 10 mM, but not affected by reducing the glucose concentration in the bath fluid or introducing physiological concentrations of inositol (30 mM) into the lumen. Secretion into the caudal lumen of unlabelled inositol measured by g.l.c. was maintained for 3 h at concentrations (300 microM) greater than those in the bath fluid and was not reduced when glucose or inositol were removed from the bath. In contrast, glucose was only detectable in the lumen when it was present in the bathing medium, reaching 1% of this concentration. Radioactivity appeared in the epididymal lumen reaching a plateau (19% of bath fluid values) in the corpus and cauda when [3H]glucose was added to the bath fluid, but no radiolabelled inositol was found in the lumen. We conclude that epididymal tissue is a major source of secreted inositol.     

Tylosin concentrations in rat serum and lung tissue after administration in drinking water

Tylosin has low in vitro minimum inhibitory concentrations against Mycoplasma pulmonis but levels attainable in rat serum or lung tissue have not been reported previously. Tylosin levels in rat serum and lung tissue were determined after administration of tylosin in the drinking water. Rats were given water mixed with a commercially available preparation of tylosin base, vitamins, and dextrose. Although the calculated amount of tylosin added to the water was intended to provide a concentration of 500 mg/L, the concentration attained was 70-79 mg/L and decreased rapidly with time. Bioassay of serum and lung tissue after 1-10 days of continuous medication revealed no detectable tylosin concentrations (less than 0.1 microgram/ml) in serum, while lung tissue from all treated rats contained tylosin (means = 10.69 +/- 2.66 micrograms/gm tissue, range = 3.93 to 18.14, n = 59). These concentrations are over ten times the reported in vitro minimum inhibitory concentrations against M. pulmonis which indicates that tylosin administration in drinking water may be useful in the treatment of M. pulmonis pneumonia in rats.     

Colorimetric analysis with N,N-dimethyl-p-phenylenediamine of the uptake of intravenously injected horseradish peroxidase by various tissues of the rat

1. A method is described for the colorimetric determination of peroxidase with N,N-dimethyl-p-phenylenediamine. The amount of red pigment formed by peroxidase is proportional to the concentration of enzyme and to the time of incubation during the first 40 to 90 seconds. The influence of the concentration of enzyme, N,N-dimethyl-p-phenylenediamine, H(2)O(2), the time of incubation, pH, the temperature, and the possible interference by oxidizing and reducing agents of tissues has been tested. 2. The method has been used to follow the uptake of intravenously injected horseradish peroxidase by 18 different tissues of the rat over a period of 30 hours. The highest concentration of the injected tracer enzyme was found in extracts of kidney, liver, bone marrow, thymus, and spleen. Considerable amounts were taken up by pancreas, prostate, epididymis, and small intestine. Lower concentrations were found in extracts of lung, stomach, heart, and skeletal muscle, aorta, skin, and connective tissue. No uptake was observed by brain and peripheral nerve tissue. 3. Tissue homogenates containing high concentrations of the injected peroxidase, in general also showed high or average activity of acid phosphatase. 4. Six hours after intravenous administration, the liver contained 27 per cent, the kidney 12 per cent, and the spleen, 1.4 per cent of the injected dose. 5. Approximately 20 per cent of the injected peroxidase was excreted in the urine during the first 6 hours, and the concentration of peroxidase in blood serum and urine fell exponentially during this time. After 6 hours, only low concentrations were excreted in the urine but low enzyme activity was still detectable after 30 hours. Approximately 6 per cent of the injected dose was excreted in the feces from 6 to 20 hours after administration. 6. After feeding through a stomach tube, low concentrations of peroxidase were found in blood serum and urine. Considerable variations in the extent of absorption from the gastrointestinal tract were observed in individual rats.     

THE NATURE AND ORIGIN OF THE SOLUBLE PROTEIN IN HUMAN AMNIOTIC FLUID

1. Amniotic fluid surrounds the human fetus and is separated from the uterus by the amnion, chorion and placenta. The ability to obtain samples of amniotic fluid from women by a simple procedure has encouraged studies on the nature and origin of the fluid, and on its use for the diagnosis of a variety of clinical conditions. The fluid contains cells, which are of fetal origin, and can be grown in a tissue culture. Cyto-genetic and biochemical analyses can therefore be used to detect chromosomal aberrations and inborn errors of metabolism in the fetus. 2. The supernatant of amniotic fluid contains many of the solutes typical of extracellular fluid. In particular, it contains a wide range of proteins and those which are of fetal origin are likely to be of use in the prenatal diagnosis of fetal disease. This review examines the nature and origin of the soluble protein in amniotic fluid, and discusses the diagnostic uses of the proteins which are of fetal origin. 3. In other mammals, the arrangement of the fetal membranes is different from that in man, and these differences are reflected by changes in the nature of the amniotic fluid. Thus data from other animals have little applicability to man. 4. Electrophoresis and immunoelectrophoresis have established that the major proteins in amniotic fluid are also present in maternal and fetal sera. Their concentrations in the fluid are influenced by their molecular weight and proteins larger than about 2.5 times 10⁶ may be excluded. Towards term, phenotyping studies show that a number of serum proteins in amniotic fluid are of maternal origin. In the case of group-specific component (Gc) this has been shown to be so throughout pregnancy. Such proteins must enter the fluid by diffusing across either the chorion or the chorionic plate and then the amnion. 5. It has been previously claimed that various serum proteins in amniotic fluid are of fetal origin. For albumin and IgG there are data that strongly support a maternal origin. The evidence on the origin of insulin is inconclusive. The concentration of β₂-microglobulin in amniotic fluid exceeds that in maternal serum and is probably too high also for fetal serum to be its major source. It has a wide tissue distribution and probably enters the fluid from surrounding structures. 6. Alpha-fetoprotein in amniotic fluid is of fetal origin as it is present in maternal serum at far lower concentrations. It is found in fetal serum, urine and yolk sac, but it is not clear how it enters the amniotic fluid of normal fetuses. The concentrations of Gc and alpha-fetoprotein have been measured in amniotic fluid and in their sera of origin. The relative concentration of Gc in amniotic fluid was found to be much greater than that of alpha-fetoprotein and the concentration gradients of these marker proteins can be compared with data for other proteins. In this way further evidence has been obtained that the albumin, α₁,-antitrypsin and transferrin in amniotic fluid are mainly of maternal origin throughout pregnancy. 7. Immunological studies have shown that at least three proteins of non-serum origin are present in amniotic fluid and they have also been located in the amnion and uterine decidua. 8. The enzymes present in amniotic fluid are summarized. Many lysosomal enzymes are clearly of fetal origin since they show altered specific activities in the appropriate cases where the fetus is affected with an inborn error of metabolism. For other enzymes, analysis of specific activity gradients can help to decide the extent to which an enzyme is of serum origin, although this will not exclude the possibility of a maternal (uterine) contribution. The results of such analyses suggest that, relative to the serum protein in amniotic fluid, the greatest concentrations of the minor non-serum proteins in the fluid occurs between thirteen and eighteen weeks of pregnancy and also towards term. 9. Some inborn errors of metabolism may be diagnosed prenatally by measuring the specific activity of the respective enzyme in amniotic fluid. However, the presence of different enzymes with similar substrate specificities has prevented this in Pompe's disease. 10. In cases where the fetus is affected with anencephaly or spina bifida there is an increase in the concentration of alpha-fetoprotein in the amniotic fluid. This has provided a way of detecting these diseases early enough to allow termination of pregnancy. 11. The discovery of new proteins in fetal serum and in the tissues surrounding the amniotic cavity would seem to provide the best chance of extending the uses of amniotic fluid into the other areas of prenatal medicine.     

Compromised antioxidant status and persistent oxidative stress in lung transplant recipients

Oxidative stress may be a key feature, and hence important determinant, of tissue injury and allograft rejection in lung transplant recipients. To investigate this, we determined the antioxidant status (urate, ascorbate, thiols and α-tocopherol) and lipid peroxidation status (malondialdehyde) in bronchoalveolar lavage (BAL) fluid and blood serum of 19 consecutive lung transplant recipients 2 weeks and 1, 2, 3, 6, and 12 months post-surgery. BAL fluid and blood samples from 23 control subjects and blood from 8 patients two days before transplantation were obtained for comparison. Before surgery, the antioxidant status of patients was poor as serum ascorbate and total thiol concentrations were significantly (p < 0.05) lower than control subjects. Two weeks post-surgery, ascorbate and total thiol concentrations were still low and urate concentrations had fallen compared to control subjects (p < 0.01). At this time, BAL fluid urate concentration was higher (p < 0.01), ascorbate concentration was lower (p < 0.01) and reduced glutathione concentrations were similar to control subjects. MDA, a product of lipid peroxidation, was higher (p < 0.01) in both BAL fluid and serum obtained from transplant patients compared to control subjects. During the first 12 months post-surgery, little improvement in antioxidant status or extent of lipid peroxidation was seen in transplant recipients. Regression analysis indicated no difference in serum or BAL fluid antioxidant status in patients with acute rejection compared to those without. In conclusion, lung transplant recipients have a compromised antioxidant status before surgery and it remains poor for at least the first year following the operation. In addition, these patients have elevated MDA concentrations in both their lung lining fluid and blood over most of this time. Oxidative stress is not, however, a sufficiently sensitive endpoint to predict tissue rejection in this group.     

Biphasic effect of gingipains from Porphyromonas gingivalis on the human complement system

Periodontitis is an inflammatory disease of the supporting structures of the teeth and is caused by, among other agents, Porphyromonas gingivalis. P. gingivalis is very resistant to killing by human complement, which is present in a gingival fluid at 70% of the serum concentration. We found that the incubation of human serum with purified cysteine proteases of P. gingivalis (gingipains) or P. gingivalis wild-type strains W83 and W50 resulted in a drastic decrease of the bactericidal activity of the serum. In contrast, serum treated with P. gingivalis mutants lacking gingipains (particularly strains without HRgpA) maintained significant bactericidal activity. To understand in detail the mechanism by which gingipains destroy the serum bactericidal activity, we investigated the effects of gingipains on the human complement system. We found that all three proteases degraded multiple complement components, with arginine-specific gingipains (HRgpA and RgpB) being more efficient than lysine-specific gingipain (Kgp). Interestingly, all three proteases at certain concentrations were able to activate the C1 complex in serum, which resulted in the deposition of C1q on inert surfaces and on bacteria themselves. It is therefore plausible that P. gingivalis activates complement when present at low numbers, resulting in a local inflammatory reaction and providing the bacteria with a colonization opportunity and nutrients. At later stages of infection the concentration of proteases is high enough to destroy complement factors and thus render the bacteria resistant to the bactericidal activity of complement.     

Influence of a new slow-release GnRH analogue implant on reproduction in the Budgerigar (Melopsittacus undulatus, Shaw 1805)

The neuroendocrine conditioning of reproduction in birds could perform a very important role in captive breeding, especially in endangered species. Whereas in domestic and wild mammals pharmacological reproductive conditioning is well developed, in birds an effective method is not available. The aim of this study was to test the influence of a new slow-release GnRH analogue (buserelin acetate) implant on the reproductive activity of the Budgerigar (Melopsittacus undulatus), used as model species for captive-bred endangered birds. The effects were assessed by looking at reproductive parameters (egg-laying rate, egg fertility rate) and measuring excreted sex steroid metabolite concentrations in male and female birds. Modification of reproductive parameters and steroid metabolites excretion patterns were observed among birds administered with a GnRH analogue implant and maintained under artificial photoperiod (group I; 16L:8D). Implanted birds showed higher rates of egg-laying, potentially a higher proportion of fertile eggs and higher excreted steroid metabolite concentrations than birds maintained under natural photoperiod (group II; 10L:14D) and birds maintained under artificial photoperiod (group III; 16L:8D). Thus, it is concluded that the new slow-release GnRH analogue implant may represent an innovative and practicable treatment to rapidly induce reproductive activity in the Budgerigar, and that excreted sex hormone metabolites detection permits to monitor male and female gonadal activity.     

Adenosine as a constituent of the brain and of isolated cerebral tissues, and its relationship to the generation of adenosine 3′:5′-cyclic monophosphate

1. Adenosine was determined in rapidly frozen rat and guinea-pig brain and in guinea-pig cerebral tissues after incubation in vitro. Adenosine concentrations were approx. 2nmol/g wet wt. in frozen tissue, diminished at room temperature, and returned to 2nmol/g on incubation in oxygenated glucose/salines. 2. Superfusion with noradrenaline then increased the tissue's adenosine concentration 2.5-fold, and hypoxia caused an 8-fold increase. 3. Electrical stimulation alone or in the presence of noradrenaline or histamine increased the tissue's adenosine and cyclic AMP, but adenosine concentrations reached their peak later and were maintained for longer than those of cyclic AMP. 4. Superfusion with l-glutamate with and without electrical excitation raised adenosine concentrations to 15-34nmol/g. The increases in cyclic AMP on electrical stimulation, superfusion with glutamate or a combination of these treatments were diminished by addition of adenosine deaminase or theophylline. 5. It is concluded that adenosine can be produced endogenously in cerebral systems, in sufficient concentrations to accelerate an adenosine-activated adenylate cyclase, and by this route can contribute to the cerebral actions of electrical stimulation and of the neurohumoral agents. In certain instances cyclic AMP as substrate contributes to an increase in adenosine.     

Resistance of Escherichia coli to penicillins. 8. Physiology of a class II ampicillin-resistant mutant

Class II ampicillin-resistant mutants of Escherichia coli are defined as having a twofold increase in penicillinase-mediated ampicillin resistance when determined by colony formation tests on plates. In this paper, one class II mutant has been compared to its parent strain. In liquid medium, the mutant was less resistant than the parent strain both in the absence and in the presence of R1 and R-factor mediating penicillinase activity. The penicillinase activity was found to be almost completely bound to the cells in the parent strain, whereas it was excreted to a great extent in the class II mutant strain. In liquid medium, resistance was well correlated to the cell-bound penicillinase activity, whereas the excreted penicillinases were also of great importance for survival on ampicillin plates. The mutant also had a changed resistance to a great number of other antibacterial drugs. The mutant was found to be more sensitive than the parent strain to osmotic shock, especially when treated with ethylenediaminetetraacetic acid or washed with sodium ions. However, the osmotic stability was restored by the presence of 1 mm Mg(2+) ions. The class II mutant was more sensitive than the parent strain to sodium cholate, and it adsorbed the phages T4 and T3-1 at a slower rate than did the parent strain. The two strains adsorbed T6 at the same rate. The class II phenotype could be gradually reversed by increasing concentrations of divalent cations. The pleiotropic changes in the phenotype are apparently unrelated to the specific targets for the antibacterial agents tested. They are secondary consequences of a cell envelope mutation. The findings indicate that the class II mutation mediates a structural change in the lipopolysaccharide of the cell envelope.     

Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3

Naturally occurring cationic antimicrobial peptides (CAPs) are an essential component of the innate immune system of multicellular organisms. At concentrations generally higher than those found in vivo, most CAPs exhibit strong antibacterial properties in vitro, but their activity may be inhibited by body fluids, a fact that could limit their future use as antimicrobial and/or immunomodulatory agents. In the present study, we evaluated the effects of human serum components on bactericidal activity of the human beta-defensin 3 (hBD-3), a CAP considered particularly promising for future therapeutic employment. Human serum diluted to 20% strongly inhibited the bactericidal activity of the peptide against both the Gram-positive species Staphylococcus aureus and the Gram-negative species Acinetobacter baumannii. Such activity was not restored in serum devoid of salts (dialyzed), pre-treated with protease inhibitors, or subjected to both of these treatments. The addition of physiological concentrations of NaCl, CaCl2, and human albumin in the bactericidal assay abolished bactericidal activity of hBD-3 against S. aureus, while it only partially inhibited the activity of the peptide against A. baumannii. Although a proteolytic activity of serum on hBD-3 was demonstrated at the protein level by Western blot, addition of physiological concentrations of trypsin to the bactericidal assay only partially affected the antibacterial properties of the peptide. Altogether, these results demonstrate a major role of mono-divalent cations and serum proteins on inhibition of hBD-3 antibacterial properties and indicate a relative lack in sensitivity of the bactericidal activity of this peptide to trypsin and trypsin-like proteases.     

Concentration compared with total urinary excretion of 11,17-DOA in cynomolgus monkey urine

Strees sensitive molecules exhibit great variation in concentration in the circulation and it may often be advantageous to quantify these in urine or feces rather than in serum or plasma. We advocate that all urine-or feces-should be collected, and that excretion of stress sensitive molecules should be expressed as amounts excreted per time unit per kg body-weight, rather than being expressed as concentrations in samples. Urine and feces excretion varies significantly within and between animals over time, which may render simple concentration measures of molecules of little biological relevance.     

Endogenous zinc concentrations in cysteamine-induced duodenal ulcers in the rat

Exogenously administered zinc compounds have been shown to possess anti-ulcer activity against a wide variety of ulcerogenic agents, both in laboratory animal models and in human peptic ulcer disease. However, a strong possibility exists that endogenous zinc may also play an important role during noxious events by various mechanisms. Therefore, the aim of this study was to focus on the changes of endogenous zinc serum and tissue concentrations in cysteamine-induced duodenal lesions. We used atomic absorption spectropho-tometry to determine the tissue and serum concentrations of zinc in normal (control) rats and those with cysteamine-induced duodenal ulcers. The results obtained in this study indicated that the onset, development and spontaneous healing of ulcer lesions were associated with certain shifts in zinc serum and tissue concentrations. Prior to ulcer formation, a significant increase was noted in serum zinc values. With the onset of duodenal lesions, zinc serum concentrations significantly decreased, while there was a significant increase in duodenal tissue concentrations when compared to healthy control animals. Zinc tissue concentrations decreased and returned to starting values by the end of the first week of spontaneous healing. This decrease in zinc tissue concentration corresponded to the healing rate of the duodenal ulcers. Serum zinc concentrations also returned to starting values within the first week period. These observations indicate and confirm that zinc could play an important role in duodenal ulcer disease and represent a natural defense system in the body.     

THE CONCENTRATIONS OF FREE AMINO ACIDS AND OTHER ELECTROLYTES IN CEREBROSPINAL FLUID,IN VIVO DIALYSATE OF BRAIN,AND BLOOD PLASMA OF THE DOG*

The concentrations of free amino acids in plasma, CSF and in vivo dialysates of peripheral blood (neck sac fluid) and central nervous tissue (brain sac fluid) from each of five dogs (neck sac fluid from four of five dogs) were determined by ion-exchange chromatography. Dialysates were obtained by implanting small dialysis sacs filled with a dextran-saline solution into the subcutaneous tissue of the neck or the parenchyma of the brain at least 10 weeks before sample collection. The mean plasma concentration of most amino acids was within the range of values reported in the literature for human or dog plasma. The concentrations of most amino acids were higher in the neck sac fluid than in plasma; this discrepancy, however, was, for the most part, small and could most likely be accounted for by falling plasma free amino acid levels prior to sample taking. Previous conclusions that the CSF concentrations of most amino acids are lower than plasma concentrations are confirmed, although the present work indicates that there may be considerable individual variation in the CSF/plasma distribution ratio with respect to most amino acids. In the brain sac fluid the concentration of nearly every amino acid was consistently higher than that in CSF and lower than that in the neck sac fluid. The potassium concentration in the brain sac fluid was significantly higher than, and the total osmolality significantly lower than, those in the neck sac fluid. On the assumption that the brain sac fluid represents a dialysate of the brain extracellular fluid, these results contradict recent findings (Bito and Davson , 1965; 1966) indicating that the potassium concentration of the cortex extracellular fluid is lower than that of ventricular or cisterna magna CSF and certainly lower than that of plasma. Because of this and on the basis of consideration of the reaction of the brain to a foreign body, the possibility that the implanted brain sac lay on the‘blood side’of the bloodbrain barrier was suggested. Some implications of this possibility are discussed.