Search PubMed⌕ Search

Biomedical subjects

J Gamble

Publications and source records attributed to J Gamble.

At least 37 records · Page 2Linked to original sources
PubMed1999

B1 integrin activation inhibits in vitro tube formation: effects on cell migration, vacuole coalescence and lumen formation.

Human endothelial cells (EC), when plated onto gels of extracellular matrix proteins such as Matrigel or collagen form capillary tubes in a process thought to mimic angiogenesis. We have shown previously that the extent of tube formation and the phenotype of the lumen are regulated by integrins (Gamble et al 1993) and lumen formation occurs through a process of vacuolization, coalescence and ultimate directional fusion of these vacuoles with the plasma membrane (Meyer 1997 et al). We now show here that activation of beta1 integrins on endothelial cells inhibits tube formation. On collagen gels, endothelial cells treated with 31 activating antibody 8A2 failed to migrate into the gel and tube formation was inhibited. Although several integrins mediate EC attachment to collagen alpha2beta1 is the chief determinant of EC behaviour since a blocking antibody to (alpha2beta1 reversed the effect of 8A2. On Matrigel tube formation was also inhibited by 8A2 treatment although cell alignment and sprout formation was still evident. Electron microscopy revealed the organisation of normal numbers of cells into solid sprouts and the formation of small intracellular vacuoles suggesting that initial stages of tube formation including cell migration were unaffected. However, beta1 integrin activation inhibited the coalescence of these small vacuoles into larger vacuoles, the recruitment of more cells into the sprout and the subsequent formation of mature lumen. The inhibition of capillary tube formation by beta1 activation was time dependent and long lasting. The critical time for activation of the beta1 integrin was the initial 1-2h after plating in order to inhibit tube formation although once activated, the beta1 mediated inhibition on Matrigel was still evident 4 days later. Our results suggest that beta1 integrins are critical in capillary tube formation in at least two phases. beta1 integrins are essential for migration of EC through collagen gels. Independently, beta1 integrins, although not involved in initial vacuole formation, are involved in the process of vacuole coalescence and subsequent lumen formation since beta1 integrin activation inhibits these processes.

Antibodies, Monoclonal↗
PubMed1998

Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase.

The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression. The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.

AMP-Activated Protein Kinases↗
PubMed1998

Changes in the arteriolar volume pulse of the finger during various degrees of tilt using near infra-red and red photoplethysmography.

1. Photoplethysmography is a widely used non invasive technique for the measurement of peripheral oxygen saturation. A more detailed analysis of the volume pulse (VP) can indicate alterations in peripheral vascular tone, due to sympathetic stimulation, stress, pain and temperature. - 2. In six healthy male volunteers we investigated changes in the VP resulting from vasoconstriction and vasodilatation induced by varies degrees of tilt. Subjects were subjected 0 degrees tilt followed by head down -8 degrees -15 degrees, -30 degrees -15 degrees, -8 degrees, 0 degrees, 15 degrees, 30 degrees , 70 degrees, 30 degrees, 15 degrees and 0 degrees. Each tilt stage was sustained for 15 minutes. Both VP - and haemodynamic changes were continuously recorded 30s before and then for 210 s after the imposition of each tilt step. We used a new computer driven soft and hardware for the analysis of the VP. 3. The VP signal was obtained with a sensor emitting 840 nm (NIR) and 640 nm (RED) light into finger tip with a sample rate of 128 Hz. All data was normalised to the initial mean value obtained at 0 degrees tilt. The signal strength parameters amplitude, and area under the curve and the first derivative of the amplitude (flux) as well as time discrete parameters, time of first maximum (Tmax), dicrote wave (Td), volume pulse decrease (Tdec) and fundamental arterial oscillation Tag = Td - Tmax were measured. 4. HR increased significantly during 30 degrees and 70 degrees tilt, but no change in the other hemodynamic parameters was observed. Amplitude, area under the curve and flux of both the Red and the NIR signal increased following head down tilt. A significant decrease of those parameters was found during foot down tilt. 5. No significant changes were found in the time discrete values, neither within each tilt step nor when compared to the initial mean value at 0 degrees tilt. 6. This study reveals that signal strength related parameters such as area under the curve, amplitude and flux reflect changes in vascular tone. Time discrete parameters however did not depict these changes and appear unsuitable for data analysis when using this specific hardware applied in the current study.

Adult↗
PubMed1998

Human calf precapillary resistance decreases in response to small cumulative increases in venous congestion pressure.

1. We studied human lower limbs to test the hypothesis that the application of small cumulative venous congestion pressure steps is associated with a reduction in precapillary resistance. 2. Strain gauge plethysmography was performed on twenty-one young subjects (22.7 +/- 0.6 years). At each of the small cumulative pressure steps, limb blood flow was estimated from the initial slope of the volume response to transient (10 s duration) elevations of venous congestion pressure to 90 mmHg, after which the congestion pressure was returned to the previous value. The blood flow at each pressure was also expressed as a percentage of the initial control value. Peak tibial arterial blood flux was assessed, in four of the subjects, using colour duplex ultrasonography and the same congestion pressure protocol. 3. We used Darcy's Law to predict the limb arterial blood flow and blood flux at each venous congestion pressure, assuming that both mean arterial blood pressure and precapillary resistance remained constant. 4. The mean +/- S.E.M. control arterial blood flow at the lowest venous congestion pressure, 4.8 +/- 0.1 mmHg, was 2.77 +/- 0.18 ml min-1 (100 ml)-1. At the highest venous congestion pressure, 59.2 +/- 0.2 mmHg, arterial blood flow was 2.45 +/- 0.35 ml min-1 (100 ml)-1 (121.6 +/- 16.9% of the initial value). This did not differ significantly from the initial control value, but was significantly greater than the predicted value of 0.77 +/- 0.13 ml min-1 (100 ml)-1 (28.6 +/- 2.1% of the initial value) calculated assuming constant resistance and sustained mean arterial pressure. The tibial arterial peak blood flux at 58.3 mmHg venous congestion pressure was 102.2 +/- 2.3% of the control value, which was significantly greater than the predicted 17.2 +/- 1.3% of control, calculated for this pressure, assuming constant resistance and sustained mean arterial pressure. 5. Our data show that lower limb arterial blood flow is sustained when venous congestion pressure is raised using small cumulative steps, even at congestion pressures approaching mean arterial blood pressure. These data support the notion that precapillary resistance is influenced by signals generated at the microvascular and post microvascular levels and transmitted via the endothelium.

Adult↗
PubMed1998

Increased microvascular water permeability in patients with septic shock, assessed with venous congestion plethysmography (VCP).

OBJECTIVES: To investigate microvascular water permeability (filtration capacity, Kf) in patients with septic and non-septic shock using a new non-invasive method for studying microvascular parameters in man. SETTING: Intensive Care Unit of a university hospital. PATIENTS AND METHODS: We investigated 28 patients, presenting with cardio-vascular instability due to either septic shock, or non-septic shock (haemorrhage, multiple trauma, respiratory and/or cardiac failure). INTERVENTIONS: We used standard invasive methods of monitoring (in-dwelling arterial lines and pulmonary artery flotation catheters) in combination with computer assisted venous congestion plethysmography (VCP) measurements, for a parallel assessment of peripheral microcirculatory parameters. RESULTS: On admission to the ICU, patients with septic shock revealed a significantly higher mean value of filtration capacity Kf = 6.1 +/- 0.4 x 10(-3) (mean value +/- standard error of the mean, ml.min-1.100 ml tissue-1.mmHg-1 = KfU) than non-septic patients Kf = 3.5 +/- 0.3 KfU (p < 0.02). The Kf values of the septic patients were significantly higher than those from age-matched patients with peripheral vascular disease (4.1 +/- 0.2 KfU, p < 0.001) and those of healthy controls (4.3 +/- 0.2 KfU, p < 0.001); the Kf values of the non-septic patients, however, were not significantly different. The highest mean Kf value observed during the stay on ICU was Kfmax 11.6 +/- 0.2 KfU in the septic group and 5.7 +/- 0.1 KfU in the non-septic group (p < 0.001). Pvi, a value reflecting the balance of hydrostatic and oncotic forces in the microcirculation, was elevated in both patient groups. On admission, in septic patients Pvi was 39.2 +/- 3.3 mmHg and in non-septic patients 35.1 +/- 2.7 mmHg, these values were not significantly different, but significantly higher than the Pvi value of healthy controls (Pvi 21.5 +/- 0.8) (p < 0.001). A weak, however significant, positive correlation was found between Kf and Pvi in both patient groups. No correlations were found between Kf, as well as Pvi, and cardiac index (CI), oxygen delivery index (DO2I), oxygen consumption index (VO2I) and systemic vascular resistance index (SVRI). CONCLUSIONS: An increase in permeability of microvessels will cause a loss of intravascular fluid and may therefore partially explain the large fluid requirements of patients in shock. It will also favour the development of oedema, which is often found in septic patients. We propose that changes in Kf are useful indices of microvascular malfunction and that VCP allows the non-invasive assessment of these parameters.

Adult↗
PubMed1998

Peripheral microvascular parameters in the nephrotic syndrome.

BACKGROUND: Peripheral edema, in combination with severe proteinuria and low serum albumin levels, is pathognomonic of the nephrotic syndrome, yet the exact mechanism of its formation is unknown. Two of the most important of the factors in Starling's forces controlling fluid filtration across the capillary have hitherto not been studied in nephrotic subjects. METHODS: The hydrostatic capillary pressure at the finger nail-fold in actively nephrotic subjects and age and sex matched controls was studied, using direct puncture of the apex of the capillary under video microscopy, and a servonulling apparatus to give a direct measurement of capillary pressure. Capillary filtration capacity (CFC) at the calf was measured noninvasively by a modern derivative of the technique of mercury strain gauge plethysmography. Fifteen nephrotic subjects with a variety of underlying pathological lesions, and age matched controls were studied. RESULTS: Contrary to the assumption of the "overflow" hypothesis of edema formation, there was no evidence of capillary hypertension. The capillary pressure showed no difference between nephrotic subjects and controls: median (range) of 17.6 (12.0 to 24.2) compared with 17.3 (9.0 to 21.6) mm Hg, P = NS. CFC was significantly higher in nephrotic subjects than controls [5.23 (3.28 to 8.52) x 10(-3) versus 3.55 (2.43 to 5.28) x 10(-3) ml/min/100 g/mm Hg, P < 0.01]. CONCLUSIONS: An increase in CFC provides a potentially novel mechanism contributing at least in part to the formation of peripheral edema in the nephrotic syndrome.

Adult↗
PubMed1998

A splice-isoform of vesicle-associated membrane protein-1 (VAMP-1) contains a mitochondrial targeting signal.

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.

Alternative Splicing↗
PubMed1997

Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31).

Tumor necrosis factor-alpha, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell-cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. In contrast, ECs failed to express E-selectin when plated on poly-L-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 +/- 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 +/- 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule-1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co- culture of subconfluent ECs with PECAM-1- coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.

Blood Platelets↗
PubMed1997

Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.

AMP-Activated Protein Kinases↗
PubMed1997

Relationship between venous pressure and tissue volume during venous congestion plethysmography in man.

1. Venous congestion strain-gauge plethysmography enables the non-invasive assessment of arterial blood flow, fluid filtration capacity (Kf), venous pressure (Pv) and isovolumetric venous pressure (Pvi) in man. One of the major assumptions of this technique, that cuff pressure (Pcuff) applied to the limb equals Pv at the level of the strain gauge, was tested in this study. 2. In nine healthy male volunteers (mean age, 29.3 +/- 1.2 years) the saphenous vein was cannulated with an 18-gauge catheter proximal to the medial malleolus. The subjects were supine and Pv was continuously measured during the application of small step (8-10 mmHg) increases in congestion Pcuff (up to 70 mmHg). Pcuff, changes in limb circumference and Pv were recorded by computer for off-line analysis. Since the determination of Kf is influenced by the changes in plasma oncotic pressure, venous blood samples were obtained at the start of the study, when Pcuff was raised to 30 mmHg and again to 65 mmHg and 4 min after deflation of the cuff. 3. The relationship between Pv and Pcuff was linear over the range of 10-70 mmHg (n = 9, 69 measurements, slope 0.91, r = 0.97, P << 0.001). The non-invasively measured calf Pv, based on the intercept of the relationship between the vascular compliance component (Va) and Pcuff, was 8.0 +/- 0.4 mmHg, which was not significantly different from the corrected invasively measured Pv value of 8.8 +/- 0.3 mmHg (P = 0.08). 4. Venous blood lactate and haemoglobin concentrations, as well as colloid osmotic pressure, total protein and albumin concentrations were unchanged throughout the protocol, whereas significant decreases in PO2 and blood glucose concentration were observed when Pcuff reached 65 mmHg. Assuming a constant oxygen consumption, this may suggest a reduction in tissue perfusion. 5. This study demonstrates the close correlation between Pcuff and Pv in the saphenous vein. Since the small congestion Pcuff step protocol does not cause significant increase in plasma oncotic pressure, we conclude that Pv, as well as Kf, can be accurately determined with this venous congestion plethysmography protocol.

Adult↗
PubMed1997

Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle.

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60-90 min) were paralleled temporally by reciprocal changes in the activity of the alpha2 but not the alpha1 isoform of 5'-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the alpha2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.

AMP-Activated Protein Kinases↗
PubMed1997

The effect of passive tilting on microvascular parameters in the human calf: a strain gauge plethysmography study.

1. Cumulative small steps in venous congestion pressure were used to study the effect of passive tilt on vascular parameters in dependent tissues. Using this protocol we have non-invasively assessed venous pressure (Pv,est), isovolumetric cuff pressure (Pv,i), which is the congestion cuff pressure (Pcuff) that has to be exceeded to induce fluid filtration. We have also assessed microvascular filtration capacity (Kf), which is the linear relationship between filtration rate (Jv) and Pcuff, when Pcuff > Pv,i, and is the product of the available exchange vessel surface area and wall conductance. 2. Subjects were passively tilted to increase the venous pressure at the level of the calf by 47.4 +/- 2.4 mmHg (mean +/- S.E.M.). The value of Pv,i increased from 20.6 +/- 1.8 to 48.5 +/- 3.8 mmHg after the imposition of the tilt. This change may reflect the increased colloid osmotic pressure at the microvascular interface that is known to occur in response to this manoeuvre. 3. The pre-tilt value of Kf did not change after the imposition of the passive tilt, the values being 3.2 +/- 0.4 x 10(-3) and 3.6 +/- 0.4 x 10(-3) ml min-1 (100 ml-1) mmHg-1, respectively, (n = 13). 4. These results support the notion that passive postural change alters the pre-capillary resistance, thereby altering the pressure and flow characteristics within the exchange vessels, but does not alter the surface area available for fluid exchange in the calf, contrary to previous findings in the dependent human foot using a single-step venous occlusion protocol.

Adult↗
PubMed1997

Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation.

Acetyl coenzyme A (CoA) carboxylase (ACC) is an important regulator of fatty acid oxidation in the heart, since it produces malonyl CoA, a potent inhibitor of mitochondrial fatty acid uptake. Under conditions of metabolic stress, 5'adenosine monophosphate-activated protein kinase (AMPK), which is highly expressed in cardiac muscle, can phosphorylate and decrease ACC activity. In this study, we determined if fatty acid oxidation in the heart could be regulated by insulin, due to alterations in AMPK regulation of ACC activity. Isolated working rat hearts were perfused with Krebs-Henseleit solution containing 11 mmol/L glucose, 0.4 mmol/L [9,10(-3)H]palmitate, and either 100 microU/mL insulin or 1,000 microU/mL insulin. Increasing insulin concentration resulted in a decrease in fatty acid oxidation rates (P < .05), a decrease in AMPK activity (P < .05), and an increase in ACC activity (P < .05) compared with the low-insulin group. A negative correlation was observed between AMPK and ACC activity (r = -.76). We conclude that insulin, acting through inhibition of AMPK and stimulation of ACC, is capable of inhibiting myocardial fatty acid oxidation.

AMP-Activated Protein Kinases↗
PubMed1997

Collection and short-term preservation of semen from free-ranging eastern grey kangaroos (Macropus giganteus: Macropodidae).

OBJECTIVES: To evaluate electro-ejaculation of free-range eastern grey kangaroos in the field and assess the efficacy of four diluents to preserve sperm motility over a 48-h period at 5 degrees C. PROCEDURE AND DESIGN: Under gaseous anaesthesia, 25 free-range kangaroos were electro-ejaculated and characteristics of the ejaculate noted. Spermatozoa obtained from eight ejaculates were diluted in phosphate buffered saline containing various combinations of egg yolk and glucose and refrigerated at 5 degrees C for 48 h. RESULTS: Spermatozoa were recovered from 24 of 28 ejaculates. Mean (+/- SEM) semen volume (mL) and pH were 25.0 +/- 1.9 and 7.1 +/- 0.1 respectively. The forward motility (%), rate of movement of sperm (0 to 5) and sperm concentration (x 10(6)/mL) were 77.4 +/- 1.5, 3.8 +/- 0.9 and 31.2 +/- 7.3 respectively. There was no significant difference between the four diluents in their ability to maintain forward motility of spermatozoa over 48 h. However, rate of movement over the same period was significantly (P < 0.01) improved when sperm were diluted in phosphate buffered saline containing 10% egg yolk. CONCLUSIONS: Electro-ejaculation is a safe and reliable method for collecting semen from free-ranging eastern grey kangaroos. Preliminary attempts at short-term preservation showed that the motility of kangaroo spermatozoa could be adequately stored for 24 h and that the addition of egg yolk to the semen diluent was beneficial for improving the rate of sperm movement.

Animals↗
PubMed1997

Upregulation of 5'-AMP-activated protein kinase is responsible for the increase in myocardial fatty acid oxidation rates following birth in the newborn rabbit.

In newborn rabbits, fatty acid oxidation rates in the heart significantly increase between 1 and 7 days after birth. This is due in part to a decrease in malonyl coenzyme A (CoA) production by acetyl CoA carboxylase (ACC). In other tissues, 5'-AMP-activated protein kinase (AMPK) can phosphorylate and inhibit ACC activity. In this study, we show that 1- and 7-day-old rabbit hearts have a high AMPK activity, with AMPK expression and activity being greatest in 7-day-old hearts. Hearts were also perfused in the Langendorff mode with Krebs-Henseleit buffer containing 0.4 mmol/L [14C]palmitate and 11 mmol/L glucose +/- 100 microU/mL insulin. In the absence of insulin, fatty acid oxidation rates were significantly higher in 7-day-old hearts compared with 1-day-old hearts. AMPK activity was also greater in 7-day-old hearts compared with 1-day-old hearts (909 +/- 60 and 585 +/- 75 pmol.min-1.mg protein-1, respectively; P < .05). In 1-day-old hearts, the presence of insulin resulted in a significant decrease in AMPK activity, an increase in ACC activity, and a decrease in fatty acid oxidation rates. In 7-day-old hearts, AMPK activity was also decreased by insulin, although ACC activity remained low and fatty acid oxidation rates remained high. Stimulation of AMPK in 7-day-old hearts with 200 mumol/L 5-amino 4-imidazolecarboxamide ribotide resulted in a further decrease in ACC activity and an increase in fatty acid oxidation rates. These data suggest that AMPK, ACC, and fatty acid oxidation are sensitive to insulin in 1-day-old rabbit hearts and that the decrease in circulating insulin levels seen after birth leads to an increased activity of AMPK. This can then lead to a phosphorylation and inhibition of ACC activity, with a resultant increase in fatty acid oxidation rates.

AMP-Activated Protein Kinases↗
PubMed1997

Changes in subcutaneous interstitial fluid pressure, tissue oxygenation, and skin red cell flux during venous congestion plethysmography in men.

OBJECTIVE: Venous congestion plethysmography enables noninvasive assessment of microvascular filtration capacity (Kf) in limbs. However, increases in fluid filtration might alter the balance of Starling forces: for example, progressive increases in interstitial fluid pressure (Pi) would reduce net fluid flux, thus underestimating Kf. Furthermore, elevation of cuff pressure to values close to diastolic blood pressure, as used in the protocol, may be itself impair tissue perfusion with unknown effects on the microvascular parameters investigated. METHODS: Pi was measured in healthy volunteers (n = 14) with a modified "Wick in needle" technique during small (8 mm Hg) cumulative increases in venous pressure (0-95 mm Hg). Changes in the hemoglobin (Hb) concentration, oxygenated hemoglobin (HbO2) concentration and oxidized cytochrome aa3 concentration were assessed in the calf using noninvasive near-infrared spectroscopy. Skin red blood cell flux close to the strain gauge was evaluated by laser Doppler fluxmetry. RESULTS: Pi at control was -0.89 +/- 0.8 mm Hg and during elevation of venous pressure remained constant until a cuff pressure of 30 mm Hg was reached. It rose thereafter to 1.57 +/- 1.3 mm Hg (mean +/- SD). Skin red cell flux was significantly reduced when cuff pressure exceeded 30 mm Hg and following cuff deflation, evidence of reactive hyperemia was obtained. Hb concentration increased significantly as a result of venous pressure elevation. No change in either HbO2 or cytochrome aa3 concentration was observed as long as cuff pressure remained under diastolic blood pressure. CONCLUSIONS: The small increase in Pi together with an absence of impaired tissue oxygenation during the venous congestion plethysmography protocol described by Gamble et al. supports the contention that this protocol enables accurate assessment of filtration capacity.

Adult↗
PubMed1997

Microvascular fluid filtration capacity (Kf) assessed with cumulative small venous pressure steps and with various degrees of tilt.

Tilt procedures are frequently used to test central and peripheral cardio-vascular reflexes. We have previously used venous congestion strain gauge plethysmography for measurement of fluid filtration capacity (Kf) in human legs and have shown that, providing small cumulative venous congestion pressure steps are applied, venous congestion pressure can be increased to arterial diastolic pressure without activating peripheral vasoconstrictor mechanisms. We have also studied the effect of passive tilting on Kf and have shown that the procedure does not influence the measured value Kf indicating that passive tilting does not after the total surface area available for fluid filtration, but rather the blood flow in the microvessels of the tissue under study. In the present protocol we compared the fluid filtration (Jv) resulting from small (7-10 mmHg) cumulative pressure steps with those obtained by altering hydrostatic load with progressive increases and decreases of head down tilt of -8 degrees -15 degrees and -30 degrees, followed by a similar pattern of 15 degrees, 30 degrees and 70 degrees of head up tilt. The values of Jv obtained in response to these procedures were compared with those deduced from the relationship between fluid filtration and venous congestion pressure (Pcuff) obtained during the small cumulative pressure step protocol. It was reasoned that reflex activation, by the tilt induced pressure load, would cause a reduction in local blood flow and enhanced microvascular fluid extraction. The resulting local increase in colloid osmotic pressure would give rise to lower values of Jv than those predicted on the basis of the Kf slope.

Adult↗