Subcellular distribution of tissue radiocopper following intravenous administration of 67Cu-labeled Cu-PTSM

The subcellular distribution of radiocopper in the brain and liver of rats has been determined following i.v. administration of Cu-PTSM, pyruvaldehyde bis(N⁴-methylthiosemicarbazonato)copper(II), labeled with copper-67. Homogenized tissue samples were separated by differential centrifugation into four subcellular fractions: (I) cell membrane + nuclei; (II) mitochondria; (III) microsomes; and (IV) cell cytosol. Upon sacrifice at 10 min post-Cu-PTSM injection, brain fractions, I, II, III and IV contain 35 ± 12, 11 ± 3, 2.8 ± 1.3 and 51 ± 7% of brain activity, respectively (n = 4). In animals sacrificed 24 h post-injection the subcellular fractions of brain tissue show little change from the radiocopper distribution seen at 10 min post-injection, although the mitochondrial fraction may contain slightly more tracer and the cytosolic fraction slightly less (I, 40 ± 10%; II, 18 ± 5%; III, 3.4 ± 1.5%; and IV, 38 ± 5%; n = 5). Subcellular fractions I, II, III and IV of liver contain 25 ± 5, 12 ± 3, 17 ± 4 and 46 ± 6% of ⁶⁷Cu tracer in animals sacrificed 10 min post-Cu-PTSM injection. An identical subcellular distribution of ⁶⁷Cu, was found in the liver following i.v. administration of ionic radiocopper (as Cu-citrate). The liver and brain cytosolic fractions at 10 min post-injection were further separated by Sephadex column chromatography. In liver cytosol, three different radiocopper components with molecular weights of about 140,000, 41,000–46,000 and 10,000–16,000 Da were found. In the brain supernatant fraction, most of the radiocopper was bound to a single low molecular weight cytosolic component (14,000–16,000 Da). These results suggest that the intracellular decomposition of tracer Cu-PTSM may result in the radiocopper entering the normal cellular pools for copper ions.     

Hypothermia, Metabolic Stress, and NMDA-Mediated Excitotoxicity

Abstract: Isolated embryonic retinas were metabolically stressed by inhibition of glycolysis either with iodoacetate (IOA) or by glucose withdrawal plus 10 mM 2-deoxy-D-glucose, and the effects of hypothermia were examined. Incubation at 30 versus 37°C during 30 min of hypoglycemia with IOA completely reduced the rapid swelling-related GABA release [6 ± 2 vs. 68 ± 10 nmol/100 mg of protein (mean ± SEM) for 30 and 37°C, respectively]. Histology of the retina immediately following 30 min of metabolic stress at 30°C appeared normal, whereas that at 37°C showed a pattern of acute edema, characteristic of NMDA-mediated acute excitotoxicity. Coincubation with a competitive or noncompetitive NMDA antagonist, respectively, CGS-19755 (10 μM) or MK-801 (1 μM), during 30 min of hypoglycemia at 37°C completely prevented tissue swelling, whereas extracellular GABA content remained at basal levels, indicating that the cytotoxic effects of IOA treatment for 30 min at 37°C were NMDA receptor mediated. Longer periods of hypoglycemia at 37° C produced acute toxicity that was only partially NMDA receptor mediated. Hypothermia delayed the onset of NMDA-mediated toxicity by 30–60 min. At 30°C, the rate of loss of ATP was slowed during the first several minutes of hypoglycemia (82 and 58% of maximal tissue levels at 30 and 37° C, respectively, at 5 min), but by 10 min, ATP levels were comparably reduced. After a transient exposure of retina to 50 μM NMDA in Mg²⁺-free medium, hypothermia significantly attenuated acute GABA release by 30%. At 24 h of recovery, lactate dehydrogenase release was decreased by 37%. Hypothermia had no effect when the exposure was done in medium containing physiological concentrations of Mg²⁺. The above results suggest that the protective effect of hypothermia during the metabolic insult is predominately directed at the cellular events that lead up to NMDA receptor involvement. Reduction in the rate of loss of ATP, however, does not fully account for the delay in involvement of NMDA receptors during metabolic stress at 30°C. The attenuation of direct NMDA-mediated toxicity in Mg²⁺-free medium further suggests that decreased temperature may result in altered channel properties during situations when the Mg²⁺ block is lifted.     

Lifetable demography and population growth of the rotifer Brachionus angularis in Kenya: influence of temperature and food density

Lifetable demography and reproductive traits of a Kenyan strain of the rotifer Brachionus angularis were investigated using individual and small batch culture approaches. The rotifer was identified morphologically before conducting studies at 20, 25 and 30 °C, using Chlorella vulgaris at 2.5 × 10⁵ to 2.5 × 10⁷ cells ml^–1. The rotifers were highly fecund, producing 2.11 ± 0.07 offspring female^–1 day^–1 and reproductive, producing 8.43 ± 0.24 offspring female^–1 at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The highest intrinsic rate of natural increase (0.74 ± 0.02 d^–1), specific population growth rate (0.49 ± 0.01), longest life expectancy at hatching (12.41 ± 0.28 d) and shortest generation time (2.87 ± 0.03 d) also occurred at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The duration of hatching to first spawning was shortest (2.86 ± 0.21 h) at 30 °C with 2.5 × 10⁷ algal cells ml^–1 and longest (8.83 ± 0.39 h) at 20 °C with 2.5 × 10⁵ algal cells ml^–1. The highest population density (255.7 ± 12.6 ind. ml^–1) was realised at 25 °C with 2.5 × 10⁶ cells ml^–1 on Day 8, whereas the lowest population density (122.0 ± 3.6 ind. ml^–1) was realised at 20 °C with 2.5 × 10⁵ cells ml^–1 on Day 8. The lorica length and width of the Kenyan strain of B. angularis are 85.6 ± 3.1 µm and 75.4 ± 3.6 µm, respectively. The rotifer optimally reproduces at 25 °C when fed with 2.5 × 10⁶ algal cells ml^–1.     

Effect of food intake on left ventricular wall stress

Objective

Left ventricular wall stress has been investigated in a variety of populations, but the effect of food intake has not been evaluated. We assessed whether left ventricular wall stress is affected by food intake in healthy subjects.

Methods

Twenty-three healthy subjects aged 25.6?±?4.5 years were investigated. Meridional end-systolic wall stress (ESS) and circumferential end-systolic wall stress (cESS) were measured before, 30 minutes after, and 110 minutes after a standardised meal.

Results

Both ESS and cESS decreased significantly (P?<?0.001) from fasting values 30 minutes after the meal, and had not returned to baseline after 110 minutes. ESS decreased from 65?±?16 kdynes/cm² (fasting) to 44?±?12 kdynes/cm² 30 minutes after, and to 58?±?13 kdynes/cm² 110 minutes after eating. cESS decreased from 98?±?24 kdynes/cm² to 67?±?18 kdynes/cm² 30 minutes after, and to 87?±?19 kdynes/cm² 110 minutes after the meal.

Conclusion

This study shows that left ventricular wall stress is affected by food intake in healthy subjects.     

Pre-harvest retting of flax with glyphosate: effect of growth stage at application on uptake, translocation and efficacy of glyphosate

Flax cv. Hera was grown in pots at a density of 2000 plants/m². At various stages of maturity plants were either sprayed with glyphosate (Roundup, Monsanto plc) at a rate equivalent to 1.4 kg a.e./ha or the uppermost leaves of the main stem were treated with ¹⁴C-glyphosate. Uniform desiccation to greater than 80% DM within 5 wk was obtained with sprays applied up to and at 2 wk after the mid-point of flowering (MP) and also at 10 wk after MP, when all seed had been shed. Sprays at 4–6 wk after MP caused little or no desiccation of the lower stem. Autoradiography demonstrated that ¹⁴C-glyphosate applied to the 10 uppermost leaves of the main stem at MP, or to the upper stem at 10 wk after MP, was distributed throughout the plant within 48 h. However, no downward translocation was detected within 48 h of applications during seed filling, i.e. at 2 or 5 wk after MP. Quantitative determinations of distribution of ¹⁴C-glyphosate were made at 48 h after application either to the five uppermost leaves on the main stem or, after leaf drop, to the base of the inflorescence. Translocation out of the treated zone was 58–67% of applied ¹⁴C-glyphosate with applications at 0, 2 or 6 wk after MP but only 13% with application to senescing leaves at 4 wk after MP. Little downward translocation occurred with any of these applications, ¹⁴C-glyphosate accumulating mainly in the upper stem and inflorescence. Thus failure of glyphosate to desiccate the lower stem may be related to poor downward translocation during seed development and to reduced uptake into, and export out of, senescing leaves.     

Regulation of catecholamine synthesis in rat brain synaptosomes

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent K_m of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10^?6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10^?7m ; noradrenaline, 25% inhibition at 5 ± 10^?6m ;and serotonin, 30% inhibition at 10^?5m . The catecholamine-induced inhibition of synthesis was antagonized by pre-incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra-synaptosomal amine levels, and that short-term exposure to depolarizing concentrations of potassium can increase synthesis.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.     

Cerebral Blood Flow and Metabolism During Hypoglycemia in Newborn Dogs

: Cerebral blood flow (CBF) and cerebral metabolic rates (CMR) were studied in newborn dogs during insulin-induced hypoglycemia. Pups were anesthetized, paralyzed, and artificially ventilated with a mixture of 70% nitrous oxide and 30% oxygen to maintain normoxia and normocarbia. Experimental animals were given regular insulin (0.3 units/gm IV); controls received normal saline. CBF was determined using a modification of the Kety-Schmidt technique employing ¹³³Xe as indicator. Arteriovenous differences for oxygen, glucose, lactate, and β-hydroxybutyrate (β-OHB) were also measured, and CMRo₂ and CMR_substrates calculated. Two groups of hypoglycemic dogs were identified; those in which blood glucose levels were greater than 0.5 mm (group 1), and those in which they were less than 0.5 mm (group 2). CBF did not change significantly from control values of 23 ± 10 ml/min/100 g (mean ±s.d. ) at both levels of hypoglycemia. Similarly, hypoglycemia did not alter CMRo₂, significantly from its initial level of 1.05 ± 0.37 ml O₂/min/100 g. Glucose consumption in brain during normoglycemia accounted for 95% of cerebral energy supply with minimal contributions from lactate (4%) and β-OHB (0.5%). During hypoglycemia, CMR_glucose. declined by 29 and 52% in groups 1 and 2, respectively, while CMR,_lactate increased to the extent that this metabolite became the dominant fuel for oxidative metabolism in brain. The cerebral utilization of β-OHB was unaltered by hypoglycemia. The findings indicate that insulin-induced hypoglycemia in the newborn dog is associated with an increase in cerebral lactate utilization, supplementing glucose as the primary energy fuel and thereby preserving a normal CMRo₂. These metabolic responses may contribute to the tolerance of the immature nervous system to the known deleterious effects of hypoglycemia.     

Modelling facilitation or competition within a root system: importance of the overlap of root depletion and accumulation zones

Background and aims

Relevant soil properties and nutrient distributions influencing crop root growth might be different under no-till (NT) and mouldboard plough (MP) management. The possible different root systems within different managements might have key impact on crop nutrient uptake and consequently crop production. Our objective was to assess the long-term combined effects of tillage and phosphorus (P) fertilization on corn (Zea mays L.) root distribution and morphology.

Methods

Corn root and soil samples were collected during the silking stage at five depths (0–5, 5–10, 10–20, 20–30 and 30–40 cm) and three horizontal distances perpendicular to the corn row (5, 15 and 25 cm) under MP and NT with three P fertilizations (0, 17.5, and 35 kg P ha^?1) for a long-term (22 years) experiment in eastern Canada. Root morphology and soil properties were determined.

Results

NT practice decreased corn root biomass by ?26 % compared to MP, mainly by decreasing the primary and secondary roots. Additionally, corn roots in NT tend to be more expansive on the surface layer with higher root length and surface densities for the depth of 0–5 cm at two sampling distances of 15 and 25 cm. The 35 kg P ha^?1 rate increased the root biomass by 26 and 41 % compared to the 0 and 17.5 kg P ha^?1 rates.

Conclusions

No-tillage practice and low rates of P fertilization reduce corn roots. This is probably caused by the weed competition in NT and the continued downward P status with low P rates over 22 years.

     

Endothelial microparticle formation in moderate concentrations of homocysteine and methionine <Emphasis Type="Italic">in vitro</Emphasis>

Microparticles (MPs) are small membrane vesicles released by stimulated or apoptotic cells, including the endothelium. Hyperhomocysteinemia (HHcy) is a blood disorder characterized by an increase in the plasma concentrations of total homocysteine (Hcy). The plasma Hcy level is determined by environmental factors (dietary habits, i.e. the intake of folic acid, FA) and genetic factors (N ⁵,N ¹⁰-methylenetetrahydro-folate reductase, MTHFR, polymorphism 677C>T). To evaluate whether moderate Hcy concentrations induce endothelial MP formation, the role of FA supplementation and the influence of MTHFR polymorphism were analysed. Human umbilical vein endothelial cells (HUVEC) were treated in vitro with 50 μM of Hcy and methionine (Met). The MP number and apoptotic phenotype were analyzed using flow cytometry. Increasing doses of FA (5, 15 and 50 μM) were used to reduce the HHcy effect. The MTHFR 677C>T polymorphism was determined. HUVEC stimulated by Hcy produced significantly more MPs than HUVEC under the control conditions: 3,551 ± 620 vs 2,270 ± 657 kMP (p = 0.02). Supplementation with FA at concentrations of 5, 15 and 50 μM reduced the MP count in the cell culture supernatant to 345 ± 332, 873 ± 329, and 688 ± 453 kMP, respectively (p = 0.03). MTHFR 677C>T heterozygosity was associated with a significant increase in MP formation after stimulation with Hcy compared to the control conditions: 3,617 ± 152 vs 1,518 ± 343 kMP (p = 0.02). Furthermore, the MTHFR genotype altered MP formation after Met loading. On average, 24% of the entire MP population was apoptotic (annexin V-positive). Endothelial function impairment due to HHcy is related to MP shedding, which may involve platelets and other blood and vascular cells. MP shedding is a physiological response to moderate HHcy.     

Increased glucose production following neurotensin administration

Neurotensin (NT), a recently isolated extract from bovine hypothalami, has been shown to have a hyperglycemic effect when injected into fed rats. This hyperglycemia has been attributed entirely to glycogenolysis, but no evidence is available regarding the effect of NT on the rate of glucose production and uptake. We have therefore used the primed-constant infusion of 6-³H-glucose technique to evaluate the effect of an intravenous injection of NT (1.2 nmole/kg) on glucose production $\text{in vivo}$ in 48-h starvèd, conscious rats (n=10). NT induced a progressive rise in plasma glucose concentration from the control value of 101 ± 2.3 to 162.2 ± 10.8 mg/dl at 30 min. Glucose production was significantly (p<.05) elevated 35–40% throughout the first 30 minutes after NT, while the rate of disappearance of glucose was slightly, but not significantly, elevated. An average of 38.2 mg/animal of glucose was required to account for the average increase in glucose production above the basal rate during the 30 minutes following NT. This requirement exceeded the total amount of glycogen (27.9 mg.) found in the livers of 48 hour starved rats. We therefore concluded that the rise in blood glucose concentration after NT injection was entirely due to an increased rate of glucose production, and that at least part of the increased glucose production represented an elevated rate of gluconeogenesis.     

Anatomical Region Differences and Age-Related Changes in Copper,Zinc, and Manganese Levels in the Human Brain

Using inductively coupled plasma-mass spectrometry after samples microwave-assisted acid digestion, zinc (Zn), copper (Cu), and manganese (Mn) levels were measured in 14 different areas of the human brain of adult individuals (n?=?42; 71?±?12, range 50–101 years old) without a known history of neurodegenerative, neurological, or psychiatric disorder. The main goals of the work were to establish the “normal” (reference) values for those elements in the human brain and to evaluate the age-related changes, a prior and indispensable step in order to enlighten the role of trace element (TE) in human brain physiology and their involvement in aging and neurodegenerative processes. Considering the mean values for the 14 regions, Zn (mean ± sd; range 53?±?5; 43–61 μg/g) was found at higher levels, followed by Cu (22?±?5; 10–37 μg/g) and Mn (1.3?±?0.3; 0.5–2.7 μg/g). The TE distribution across the brain tissue showed to be quite heterogeneous: the highest levels of Zn were found in the hippocampus (70?±?10; 49–95 μg/g) and superior temporal gyrus (68?±?10; 44–88 μg/g) and the lowest in the pons (33?±?8; 19–51 μg/g); the highest levels of Cu and Mn were found in the putamen (36?±?13; 21–76 μg/g and 2.5?±?0.8; 0.7–4.5 μg/g, respectively) and the lowest in the medulla (11?±?6; 2–30 μg/g and 0.8?±?0.3; 0.2–1.8 μg/g, respectively). A tendency for an age-related increase in Zn and Mn levels was observed in most brain regions while Cu levels showed to be negatively correlated with age.     

Mechanism for the increase in plasma renin activity by pentobarbital anesthesia

The mechanism by which pentobarbital anesthesia causes increases in plasma renin activity (PRA) was examined in dogs infused with either propranolol or indomethacin, an inhibitor of prostaglandin synthetase. Infusion of propranolol at 1 mg/kg, (I.V.) followed by 0.6–0.7 mg/kg/hr decreased PRA from 6.98±2.49 ng/m1/hr during control periods to 1.58±0.79 ng/m1/hr 30 minutes after the injection of propranolol (P<0.025). Subsequent induction of anesthesia with sodium pentobarbital caused PRA to rise to 3.87±0.93 ng/m1/hr in 30 minutes. (P<0.01). Plasma potassium concentration decreased from 4.6±0.2 mEq/L to reach 4.0±0.1 mEq/L 30 minutes after induction of anesthesia (P<0.005). Infusion of indomethacin at 5 mg/kg, (I.V.) followed by 1.5 ? 3.1 mg/kg/hr into conscious dogs did not decrease PRA. In contrast to the report by Montgomery et al (Fed. Proc. 36: 989, 1977), we found that the increase in PRA after pentobarbital anesthesia could not be blocked by indomethacin. PRA was 5.3±1.2 ng/m1/hr(M ± SEM) during control periods and was 4.7±1.4 ng/m1/hr 30 minutes after the infusion of indomethacin (P<0.1). PRA increased to 10.9±2.3 ng/m1/hr, 9.2±2.2 ng/m1/hr, and 7.7±1.7 ng/m1/hr at 5, 15 and 30 minutes, respectively, after the administration of pentobarbital (P<0.005, P<0.025, P<0.05). PRA declined to 4.2±1.3 ng/m1/hr 60 minutes after pentobarbital anesthesia (P<0.1). It is concluded that the mechanism by which pentobarbital causes increases in PRA is independent of prostaglandins.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Homologous priming in chemotactic peptide-stimulated neutrophils

The kinetics and dose-dependence of activation of human neutrophils exposed to sequential additions of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP) have been investigated by multiwell microplate assays. Treatment of neutrophils with medium–high doses (from 10^?8 to 5 × 10^?7 M) of fMLP caused activation of superoxide anion (O) production, but prevented further activation by a subsequent addition of an optimal dose (from 10^?7 M to 5 × 10^?7 M) of fMLP. These findings represent an example of cell desensitization, or adaptation. However, neutrophils treated with low, sub-stimulatory doses (from 10^?10 to 5 × 10^?9 M) of the peptide and then treated with optimal doses of fMLP exhibited an O production that was two to three-fold higher than that induced by the same optimal doses on untreated cells. A similar phenomenon of homologous priming of the oxidative metabolism of neutrophil has not previously been described or characterized. Priming was maximal after about 30 min of incubation with fMLP, which differed from desensitization, which required only a few minutes. Homologous priming was not confined to O production, but was also observed with the release of the granule enzyme, lysozyme. Low doses of fMLP were also capable of triggering an increase of intracellular free Ca²⁺ and of fMLP membrane receptors, which are possible mechanisms responsible for priming.     

The effect of 2-deoxyglucose on guinea pig polymorphonuclear leukocyte phagocytosis

The effects of 2-deoxyglucose (DOG), an inhibitor of glycolysis, on guinea pig polymorphonuclear leukocytes (PMN) obtained from peritoneal exudates was examined. ATP levels in PMN were reduced by 40% by one hour following an incubation with 2-deoxyglucose. When complement (C3) coated ¹⁴C-staphylococcus aureus, C3 coated lipopolysaccharide-paraffin oil droplets (LPSPO), ¹⁴C-pneumococcus opsonized with IgG, or albumin coated paraffin oil droplets opsonized with IgG were added to cell suspensions containing DOG, the phagocytizing rate was 1,310 ± 55 cpm/5 x 10⁶ cells/15 minutes, 6 ± 2 μg paraffin oil (PO)/10⁷ cells/minute, 2,250 ± 175 cpm/1 x 10⁶ cells/20 minutes or 0.037 ± 0.01 mg PO/10⁷ cells/minute compared to control values of 5,970 ± 275 cpm/5 x 10⁶ cells/15 minutes, 35 ± μg PO/10⁷ cells/15 minutes, 4,510 ± 200 cpm/1 x 10⁶ cells/20 minutes and 0.067 ± 0.01 mg PO/10⁷ cells/minute. In parallel studies the phagocytic index for latex was 0.74 ± 0.28 in DOG compared to control of 2.36 ± 1.13 and the phagocytic rate of albumin coated paraffin oil droplets was 0.029 ± 0.01 mg PO/10⁷ PMN/minute in DOG compared to control of 0.048 mg PO/10⁷ cells/minute. When ATP levels were maintained by the simultaneous addition of 5 mM glucose or pyruvate to media containing DOG, latex ingestion was improved to 1.15 ± 0.3 with glucose and 1.59 ± 0.64 with pyruvate and albumin coated particles to 0.045 ± 0.01 mg PO/10⁷ PMN/minute with pyruvate. There was no improvement in the uptake of either the C3 dependent particles or IgG coated Pneumococci in media containing DOG and glucose and/or pyruvate. Following the removal of DOG from the extracellular medium and the addition of pyruvate or glucose, phagocytosis of C3 dependent LPS-PO was restored to normal values. Neither the binding of C3 or IgG coated particles to the PMN nor the lateral movement of glycoprotein utilizing concanavalin A capping was affected by DOG. Thus, the presence of DOG in the PMN containing adequate amounts of ATP will selectively and reversibly inhibit those surface events required for phagocytosis of C3 and IgG bound particles but not latex particles or albumin particles which non-specifically bind to PMN.     

Effect of liposomally encapsulated MTX-DMPE conjugates upon TNFα and PGE2 release by lipopolysaccharide stimulated rat peritoneal macrophages

The ability of liposomally encapsulated preprations of methotrexate (MTX) and three of its lipophilic derivatives (MTX-γ-DMPE, MTX-α-DMPE and MTX-α,γ-diDMPE) to alter mediator release by lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages (PMΘ) was investigated. The viability of these macrophages when incubated with approximately 6.0 nmol/10⁵ cells of the respective liposomal preparations (MTX-LIPO, MTX-γ-LIPO, MTX-α-LIPO and MTX-di-LIPO) for 20 h was greater than 80%. Treatment of macrophages, which had been incubated with MTX-α-LIPO (5.5 nmol/10⁵ cells), MTX-γ-LIPO (6.9 nmol/10⁵ ± 9.6%, 80.6 ± 5.6% and 91 ± 11.4% phagocytosis respectively (mean ± S.E.M.). At similar concentratio MTX-α-LIPO MTX-γ-LIPO and MTX-di-LIPO (6.5 nmol/10⁵ cells), PGE₂ release from LPS-stimulated rat peritoneal macrophages was inhibited by 85.3% ± 3.7%, 68.7 ± 0.6% and 88.8 ± 2.2% respectively (mean ± S.E.M., n = 4). Incubation of these macrophages with 12, 10 and 9.4 nmol/10⁵ cells of the respective liposomal preparations resulted in 89 ± 3.3%, 62 ± 5.5% and 85 ± 3.9% inhibition of TNF_α release (rmmean ± S.E.M., n = 4). However, at this concentration MTX-di-LIPO was toxic. Neither MTX (20?2.5 nmol/10⁵ cells) nor MTX-LIPO (5.6 nmol/10⁵ cells) affected TNF_α release from LPS-stimulated macrophages. Whilst free MTX wasl also ineffective at inhibiting PGE₂ from these cells, incubation with MTX-LIPO at the above concentration resulted in 76.9 ± 2.6% inhibition of the prostaglandins release.     

Rapid block of gonadotropin uptake by corpora lutea in vivo induced by prostaglandin F2α

Intravenous administration of ¹²⁵I-hCG to 7–8 day pseudopregnant rats resulted in maximum uptake of radioactivity to corpora lutea 2 hours after treatment. At this time tissue/plasma radioactivity ratios on an equal weight basis were: corpora lutea, 70.2 ± 12.8; ovarian interstitium, 4.6 ± 0.2; kidney, 2.2 ± 0.1. No appreciable uptake was seen by adrenals or liver. Radioactivity in corpora lutea was associated primarily with membranes which sedimented at 2000g and when released by heat it was more than 63% bound to luteal LH receptor preparation in vitro. Radioactivity in renal tissue was associated primarily with the 100,000g supernatant fraction and was bound less than 1% to luteal LH receptors in vitro.PGF₂α significantly reduced uptake (p<.001) of ¹²⁵I-hCG by corpora lutea within 30 minutes (?63%) as well as at 1 (?64%), 2 (?75%), 4 (?68%) and 24 hours (?85%). No clear effect of PGF₂α on uptake of ¹²⁵I-hCG by ovarian interstitial tissue was seen. Plasma progesterone was significantly decreased (p<.001) within 30 minutes (?47%; p<.01) after PGF₂α treatment and also at 1 (?65%), 2 (?82%), 4 (?68%) and 24 hours (?92%). Two hours after PGF₂α treatment the content of progesterone in corpora lutea was depressed (?46%; p<.001). It is suggested that the rapid inhibition of luteal progesterone production induced by PGF₂α in vivo occurs through a block in gonadotropin uptake by corpora lutea.     

Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages and chlorine

Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10⁷ PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10⁷ PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre‐existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre‐existing biofilms. However, a combination of phages (3 × 10⁷ PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one‐time treatment at the concentration of 1.9 × 10⁸ PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10⁵ PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces. Biotechnol. Bioeng. 2013; 110: 286–295. © 2012 Wiley Periodicals, Inc.     

PP and PT Intervals and TP Segment in Human Electrocardiogram under Experimental Acute Normobaric Hypoxia

Seven male volunteers aged 22–27 years took part in the laboratory experiment for the determination of body response to acute normobaric hypoxia where they inhaled an oxygen–nitrogen gas mixture (9% of О₂) for 25 min. At least 100 cardiac cycles at each of six experimental time points have been recorded by using an electrocardiogram (ECG) recording technique in standard lead II. Mean heart rate (±SD) before hypoxia was 64 ± 2 bpm; the duration of the PP interval was 0.94 ± 0.07 s, that of the PT interval was 0.51 ± 0.02 s, and that of the TP segment was 0.41 ± 0.07 s. Five minutes after hypoxia, it has been found that heart rate (HR) increased by 19%; the duration of the PP and PT intervals and TP segment decreased by 16%, 6%, and 30%, respectively. Twenty minutes after hypoxia, all the parameters reached their initial values. Five and fifteen minutes after hypoxia, HR reached 59 ± 3 bpm; PP interval and TP segment increased by 9% and 14%, respectively, compared to the initial values; PT interval was the same as the initial value at the baseline. The correlation coefficient r_p (PP/PT) was 0.10–0.54; r_p (PP/TP) was 0.94–0.99. The intervals PP and TP were found to be identical.