Patterns of clometacillin circulation in the body of laboratory animals]

Distribution of clometacillin in mice, rats and rabbits was studied in comparison with some other penicillins. It was found that clometacillin was superior to all other penicillins used for comparison with respect to the circulation time in the blood after intravenous, intramusclar or intrastomach administration. As for the capacity for penetrating into the tissues from the blood, clometacillin was not inferior to ampicillin and benzylpenicillin and was superior to propicillin, though it was bound by the blood serum proteins to a greater extent than the other penicillins used for comparison.     

A pilot to examine the logistical and feasibility issues in testing deceased tissue donors for vCJD using tonsil as the analyte

Transplanted tissues have transmitted transmissible spongiform encephalopathies and in the UK there have been more cases of variant Creutzfeldt-Jakob disease (vCJD) than elsewhere in the world. A pilot study was undertaken to look at the feasibility of testing for vCJD in deceased donors using tonsillar tissue. This pilot showed that obtaining consent for removal and testing tonsil tissue was feasible. Donor eligibility for inclusion in the pilot was limited to tissue donors from the National Health Service Blood and Transplant, Tissue Services and to donors shared with the Corneal Transplant Service Eye Banks. Obtaining tonsillar tissue in the immediate post–mortem period was limited by the presence of rigor mortis. Tonsillar tissue was suitable for routine analysis for the presence of prion associated with vCJD in deceased tissue donors. Production and processing of tissue was straightforward and a low assay background was obtained from most samples. Since palatine and lingual tonsil tissue can be obtained in pairs it was possible, in the majority of cases, to set aside an intact sample for confirmatory testing if required. In one instance a sample was reactive by Western blot. However, the pattern of reactivity was not typical for that obtained from vCJD patients. Unfortunately the sample was not of sufficient quality for the confirmatory test to provide a conclusive result.     

Azaperone and azaperone-ketamine as a neuroleptic sedative and anesthetic in rats and mice

Azaperone alone and combined with ketamine were evaluated as sedative and anesthetic agents in outbred rats and mice. Using azaperone alone the duration of immobility was 1.9 to 10.8 hours for mice and 0.9 to 2.4 hours for rats. The withdrawal reflex was not eliminated from mice receiving azaperone alone; however, the withdrawal reflex was eliminated from 0.9 to 2.4 hours in rats receiving azaperone. Azaperone produced a tachypnea in rats and male mice while a depressed respiratory rate was observed in female mice. Using azaperone combined with ketamine, the duration of immobilization was 1.1 to 8.8 hours for mice and 1.3 to 6.0 hours for rats. The duration loss of the withdrawal reflex, which was used as an indication of surgical anesthesia, was 0.9 to 1.8 hours for mice and 1.0 to 6.0 hours for rats. An increase in respiratory rate was observed in rats given the combination while mice given the combination showed transient tachypnea followed by bradypnea. Overall, azaperone alone was shown to provide sedation in mice as compared to a dose dependent anesthesia in rats. The azaperone-ketamine combination produced a surgical plane of anesthesia in both rats and mice. Azaperone and the azaperone-ketamine combination appear to be a suitable alternative to sedatives and anesthetics currently used in rats and mice.     

Alginate/carbon composite beads for laccase and glucose oxidase encapsulation: application in biofuel cell technology

Alginate–carbon beads were prepared in order to develop a biocompatible matrix for laccase and glucose oxidase immobilization for application in biofuel cell technology. The enzyme loading capacity was high (91%) in pure alginate beads for glucose oxidase. For laccase, the loading capacity was enhanced from 75% to 83% by introducing carbon. Desorption out of the matrix was controlled by the enzymes’ diffusion and reached a plateau after 40 h for laccase and 70 h for glucose oxidase. Two-thirds of both enzymes was irreversibly retained inside the alginate beads. This proportion increased to 80% for laccase in combined alginate/carbon beads. Half-life of the adsorbed enzyme was enhanced to 74 days for laccase in carbon/alginate beads and 45 days for glucose oxidase in pure alginate as compared to 38 days and 23 days for free enzymes, respectively.     

Glucose and glutamine utilization by rat lymphocytes, monocytes and neutrophils in culture: a comparative study

Glucose and glutamine utilization and production of glutamate and lactate were determined for up to 48 h in lymphocytes, monocytes and neutrophils cultured in medium rich in metabolites and vitamins. Glucose was utilized by the three cell types in culture in the following order: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the order: monocytes > neutrophils > lymphocytes. The consumption of glucose followed the activity of glucose-6-phosphate dehydrogenase but it was not related to hexokinase activity. Glutamine was consumed by the three leukocyte types in culture as follows: neutrophils > lymphocytes > or = monocytes. The consumption of glutamine was not fully related to the activity of phosphate-dependent glutaminase. The production of glutamate was not remarkably different among the three cell types. For comparison, glutamine and glucose utilization and glutamate and lactate production were also evaluated using 1-h incubated leukocytes. Under this condition, only glucose or glutamine was added to the medium. Glucose was utilized as follows: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the following order: monocytes > or = neutrophils > lymphocytes. Glutamine was consumed as follows: neutrophils > lymphocytes > monocytes, whereas glutamate was produced as follows: neutrophils > or = monocytes = lymphocytes. The ratio of the amount of glucose/glutamine consumed by 1-h incubated cells was 0.5 for neutrophils, 1.5 for monocytes, and 0.3 for lymphocytes. However, the three cell types cultured for 48 h utilized glucose to a much higher degree than glutamine. The ratio of the amount of glucose/glutamine utilized by the cultured cells was 8.9 for neutrophils, 16.4 for monocytes, and 6.7 for lymphocytes. These observations support the proposition that glutamine is required in much higher amounts than glucose to accomplish the total metabolic requirement of leukocytes. Under conditions closer to physiological when the availability of a variety of metabolites and vitamins is not restricted, glucose is the preferred substrate for lymphocytes, monocytes and neutrophils.     

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.     

Leaf habit influences nitrogen remobilization in Vaccinium species.

The effect of N supply on plant growth and leaf demography of a deciduous and an evergreen Ericaceae was studied in relation to their internal cycling of N. Mature ramets of Vaccinium myrtillus (deciduous) and Vaccinium vitis-idaea (evergreen) were established in sand culture for 1 year with an adequate supply of a balanced nutrient solution. During one growing season, the plants were given two levels of N supply enriched with 15N and eight sequential destructive harvests were taken. Recovery of unlabelled N in the new shoot was used to determine the remobilization of N from storage. Initially, growth was unaffected by N supply. After May, High N enhanced growth for both species but the nature of their growth response differed. For both species, new shoot biomass and leaf number increased but root biomass production was affected for V. myrtillus only. Whole plant biomass production was similar for both species under High N, but was greater for V. vitis-idaea under Low N. The amount of N remobilized to support new shoot growth was similar for the two species and was independent of N current supply. N was remobilized predominantly from previous year leaves for V. vitis-idaea and from previous year stems and roots for V. myrtillus. The contribution of remobilization to new shoot N was similar for the two species, but depended on N supply. Remobilization was faster in V. myrtillus, but lasted longer in V. vitis-idaea. The results are discussed in relation to species growth in N-poor environments, focusing on the extent to which species-differences in the dynamic of N remobilization and growth may explain their adaptation to constant and/or changeable N supply.     

Cross prediction studies on spring barley

Summary The genetical and environmental control of three height characters, two maturity characters and neck length in five barley pair crosses was studied using both F₂ triple test cross and model fitting analysis.Significant additive and dominance effects were found for all six characters with some evidence of epistasis for each character. Generally, dominance was incomplete for the height characters but was significantly directional for increased height in those crosses where dwarfing genes were segregating. Variable dominance effects were found for both the maturity characters. Complete dominance was found in three cases, otherwise incomplete dominance was found. Significant directional dominance for earliness was found for both maturity characters in one cross but this was attributed to the presence of a daylength insensitivity factor in one of the parents. Most of the genetic variation for neck length was additive, though some evidence of dominance was found.Broad sense and narrow sense heritability estimates generally were found to be high for the height and maturity characters but low for neck length. It was concluded that early generation selection for height at ear emergence, for final height and for awn emergence was worthwhile. Early generation selection for neck length was not recommended from the results of this study.     

Leaf hydraulic conductivity and stomatal responses to humidity in amphistomatous leaves

The response of stomata to changes in humidity for a single surface of an amphistomatous leaf was investigated in Xanthium strumarium and Vicia faba using gas exchange and direct observation of stomatal apertures. The stomatal response to humidity for a given surface was found to be the same whether or not the humidity for the opposite surface was changed concurrently. Stomata on the surface for which humidity was constant showed no response to changes in humidity for the opposite surface. Despite large changes in epidermal turgor on the surface for which humidity was changed, there was no change in epidermal turgor for the surface with constant humidity. Measurements of transpiration and epidermal turgor as functions of the mole fraction gradient of water between leaf and air were used to calculate a value for leaf hydraulic resistance. The results suggest that in these species, the mechanism for the stomatal response to humidity resides in the epidermis or the mesophyll very close to the epidermis, and that most of the hydraulic resistance of the leaf occurs between the xylem and the evaporating sites.     

Measurement of blood-brain barrier permeation in rats during exposure to 2450-MHz microwaves

Adult rats anesthesized with pentobarbital and injected intravenously with a mixture of [¹⁴C]sucrose and [³H]inulin were exposed for 30 min to an environment at an ambient temperature of 22, 30, or 40 °C, or were exposed at 22 °C to 2450-MHz CW microwave radiation at power densities of 0, 10, 20, or 30 mW/cm². Following exposure, the brain was perfused and sectioned into eight regions, and the radioactivity in each region was counted. The data were analyzed by two methods. First, the data for each of the eight regions and for each of the two radioactive tracers were analyzed by regression analysis for a total of 16 analyses and Bonferroni's Inequality was applied to prevent false positive results from numerous analyses. By this conservative test, no statistically significant increase in permeation was found for either tracer in any brain region of rats exposed to microwaves. Second, a profile analysis was used to test for a general change in tracer uptake across all brain regions. Using this statistical method, a significant increase in permeation was found for sucrose but not for inulin. A correction factor was then derived from the warm-air experiments to correct for the increase in permeation of the brain associated with change in body temperature. This correction factor was applied to the data for the irradiated animals. After correcting the data for thermal effects of the microwave radiation, no significant increase in permeation was found.     

Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.     

Feeding, defecation, and development times of Meccus longipennis Usinger, 1939 (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Aspects related to hatching, time-lapse between presenting the blood-meal and beginning of feeding, feeding time, postfeed defecation delay, mortality, and fecundity for each stage of Meccus longipennis life-cycle were evaluated. The bugs were maintained in a dark incubator at 27 1 C and 80 5% rh, were fed weekly and checked daily for ecdysis or death. The hatching rate observed for 300 eggs was 76.7% and the average time of hatching was 19.8 days. Mean time-lapse between presentation of the blood meal and the beginning of feeding was under 5 min in nymphal stages and postfeed defecation delay was under 10 min in most stages, except in fourth and fifth stages. Mean feeding time was longer than 10 min in most stages, except in fourth stage. One hundred thirty-one nymphs (N) (65.5%) completed the cycle and the average time from NI to adult was 192.6 34.8 days. The average span in days for each stage was 18.1 for NI, 21.4 for NII, 29.5 for NIII, 45.5 for NIV and 55.9 for NV. The number of bloodmeals at each nymphal stage varied from 1 to 5. The mortality rate was 3.29 for NI, 6.8 for NII, 2.92 for NIII 3.76 for NIV, and 10.16 for NV nymphs. The average number of eggs laid per female in a 9-month period was 615.6. Based on our results, we conclude that M. longipennis has some biological and behavioral characteristics which influence its capacity of becoming infected and transmitting Trypanosoma cruzi to human populations in those areas of Mexico where it is currently present.     

Biology of Triatoma pallidipennis stal 1945 (Hemiptera: Reduviidae:Triatominae) under laboratory conditions

Aspects related to hatching, life time, mortality, feeding behaviour and fecundity for each stage of Triatoma pallidipennis life-cycle were evaluated. The hatching rate observed for 200 eggs was 60% and the average time of hatching was 18 days. Eighty nymphs (N) (40%) completed the cycle and the average time from NI to adult was 168. 7+/-11.7days. The average span in days for each stage was 18.0 for NI, 18.5 for NII, 30.0 for NIII, 35.7 for NIV and 50.1 for NV. The number of bloodmeals at each nymphal stage varied from 1 to 5. The mortality rate was 9.17 for NI, 5.5 for NII, 6.8 for NIII 4.17 for NIV and 13.04 for NV nymphs. The average number of eggs laid per female in a 9-month period was 498.6. The survival rates of adults were 357+/-217.9 and 262.53+/-167.7 for males and females respectively.     

Viscoelasticity of fibrinogen solution and of blood during coagulation studied by a new damped oscillation rheometer

The behavior of a newly developed damped oscillation type rheometer was analyzed for fibrinogen solution and blood during coagulation. This rheometer consists of a cylindrical tube suspended from a torsion wire, that is filled with liquid to be tested. The logarithmic damping factor (LDF) during coagulation for blood and fibrinogen solution was obtained by this rheometer, which was closely related to the changes of viscosity and/or viscoelasticity of the blood sample. The slight increase of LDF prior to the rapid decrease was observed for blood. The increase of LDF would be reflected in the formation of the aggregation structure of red blood cells (rouleaux network) prior to the formation of fibrin network. The value of LDF for fibrinogen solution sharply increased and then decreased through a maximum value with the progress of coagulation, although the change of LDF was remarkably dependent on the fibrinogen concentration. The initial increase in LDF for fibrinogen solution was considered to be due to the formation of small clots in the solution. The decrease in LDF after attaining a maximum value is ascribed to the formation of fully developed fibrin network. The maximum value of LDF during coagulation for fibrinogen solution is higher than that for blood. The behavior was compared with that for non-biological fluids such as viscosity standard liquids and polyvinyl alcohol solution. From those data, it was concluded that the higher value of LDF than that for Newtonian liquids was due to the formation of aggregation structure or inhomogeneous fine clots in the liquid, which was accompanied with the appearance of the elasticity.     

The RBE of neutrons for induced mitotic gene conversion in "error-prone repair" defective yeast

Mitotic gene conversion was induced in the diploid yeast strain D7.rad6 which lacks "error-prone repair" and thus does not mutate. Neutrons (14.5 MeV), 60Co gamma rays, and 150 kVp X rays delivered under oxic or anoxic conditions were compared for their ability to induce gene conversion. Doses were chosen to minimize cell killing. A lack of induced mutation in this strain at the ilv1-92 allele was confirmed. Gene conversion of the trp5-27/trp5-12 alleles was induced with a linear dose response, and the yield of convertants per gray was significantly enhanced over yields reported previously for a wild-type stain. The relative biological effectiveness (RBE) of neutrons relative to low-LET radiations was found to be about 2.2 for either oxic or anoxic radiation in contrast to wild-type where the oxic RBE was 1.7 and the anoxic RBE 2.7. Absence of the rad6 function was therefore associated with an altered RBE for the conversional end point. The oxygen enhancement ratio (OER) for gene conversion was found to be about 1.7 for all radiations in contrast to the wild type where the OER for neutrons was 1.7, but for low-LET radiations it was 2.7. As repair of ionizing damage in the rad6 strain did not lead to mutation, owing to the loss of "error-prone repair," the changes in yield, RBE, and OER were consistent with the hypothesis that some of the lesions processed by wild type to generate mutations could, in the rad6 strain, lead instead to gene conversion.     

Brain CCK receptors: species differences in regional distribution and selectivity

The binding of 125I-CCK-33 to its receptors prepared from cerebral cortex and cerebellum was studied in four species: mouse, rat, hamster, and guinea pig. Only the guinea pig showed significant binding to membranes from cerebellum and this binding was comparable to that observed for cerebral cortex. In all four species, the order of potency of unlabeled analogs to compete for the binding site was CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. While the affinity for CCK-8 and CCK-33 was similar in the various species, the relative affinity for desulfated CCK-8 and CCK-4 was less for hamster and guinea pig, indicating species differences in receptor specificity, as well as in regional localization.     

The effect of bovine serum albumin on partial reactions of palmitoyl-CoA chain elongation by rat liver microsomes.

In the absence of albumin, v/s curves for both condensation and overall chain elongation demonstrated that the specific activity for overall chain elongation was 3.7 times that of condensation. When the molar ratio of palmitoyl-CoA to albumin was greater than 2 : 1, the specific activity of chain elongation exceeded that of condensation. At these low albumin concentrations, in the absence of NADPH, the beta-ketostearoyl-coA was converted back to palmitate. This cleavage reaction is inhibited by albumin in a concentration-dependent manner. When the palmitoyl-CoA to albumin molar ratio was less than 2 : 1, the specific activity for condensation exceeded that for overall chain elongation and some beta-ketostearate was shown to accumulate under chain elongation conditions. The specific activity for dehydration of beta-hydroxystearoyl-CoA was maximal when the acyl-CoA to albumin molar ratio was between 10 : 1 and 4 : 1 but the rate of this reaction was not markedly influenced by variations in albumin concentration. The specific activity for the NADPH-dependent reduction of 2-trans-octa-decenoyl-CoA was 18 nmol . min(-1) . mg(-1) in the absence of albumin and increased to a maximum of 112 when the substrate to albumin molar ratio was 2 : 1. At higher albumin concentrations the reductase reaction was inhibited. Conversely, the specific activity for the reverse dehydrase was maximal at low albumin concentrations and the rate of this reaction declined as the albumin concentration increased. Our results demonstrate that albumin not only alleviates a substrate induced inhibition but also regulates the metabolic fate of 2-trans-octadecenoyl-CoA and in this regard may possibly substitute for acyl-CoA binding proteins.     

Sensitive high-performance liquid chromatographic fluorescence assay for the quantitation of topotecan (SKF 104864-A) and its lactone ring-opened product (hydroxy acid) in human plasma and urine

A sensitive reversed-phase high-performance liquid chromatographic fluorescence method is described for the simultaneous determination of topotecan (I) and the hydrolysed lactone ring-opened product hydroxy acid (II) in plasma and for the determination of I in urine. To 250 μl of plasma, a 750-μl volume of cold methanol was added to stabilize the pH-dependent conversion of I into II. In plasma, the lower limit of quantitation (LLQ) for both compounds was 0.10 ng/ml. The between-day variation for I at the LLQ was 7.1% and for II was 5.5%. Prior to injection, urine samples were acidified with orthophosphoric acid and diluted with phosphate-buffered saline (PBS). In urine, the calibration curve for I was linear in the range of 10 to 250 ng/ml and the LLQ was 10 ng/ml. The assay was developed to enable pharmacological analysis of I, in on-going phase I and II studies, in patients with solid tumors.     

Oxidation of D-lactate and L-lactate by Neisseria meningitidis: purification and cloning of meningococcal D-lactate dehydrogenase.

Neisseria meningitidis was found to contain at least two lactate-oxidizing enzymes. One of these was purified 460-fold from spheroplast membranes and found to be specific primarily for D-lactate, with low-affinity activity for L-lactate. The gene for this enzyme (dld) was cloned, and a dld mutant was constructed by insertional inactivation of the gene. The mutant was unable to grow on D-lactate but retained the ability to grow on L-lactate, providing evidence for a second lactate-oxidizing enzyme with specificity for L-lactate. High-affinity L-lactate-oxidizing activity was detected in intact bacteria of both the dld+ and dld mutant strains. This L-lactate-oxidizing activity was also seen in sonicated bacteria but was reduced substantially on detergent solubilization or on preparation of spheroplast membranes.     

Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L.

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.