A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20.

Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCR TaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838-843]. The pCS20 quantitative real-time PCR TaqMan probe was compared to the currently used pCS20 PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCR TaqMan probe was the most sensitive assay detecting seven copies of DNA/mul of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCR TaqMan probe assay was the most sensitive and can be performed within 2h it is an effective assay for epidemiological surveillance and monitoring of infected animals.

In vivo heat shock protects rat myocardial mitochondria.

Heat shock (HS)/stress proteins (HSP) provide protection from a variety of stresses other than HS, including oxidative stress and mitochondria have been implicated as the target of HS-related protection in stressed cultured cells. Here we investigated whether mitochondria also are targets for the HS-mediated protection in vivo. Sprague Dawley rats were exposed, or not, to HS (41 degrees C, 15 min). After a 21 h recovery period, hearts were excised and perfused with or without H2O2 (0.15 mM). Myocardial mitochondria were then isolated, and their oxygen consumption was analyzed. HS prevented H2O2-induced alterations in state 3 respiration while increasing the expression of Hsp70 and heme oxygenase (HO). Thus, in vivo HS protects rat myocardial mitochondrial respiration against the deleterious effects of oxidative injury, a protection relating to Hsp70 and/or HO and targeting state 3 respiration.

Two roles for caveolae in the cardiac myocyte?

The presence of caveolae in many cell types including heart myocytes is well established. It is hypothesized that caveolae may play a role in the storing of excess Ca2+ and may be instrumental in Ca2+ transients during contraction and relaxation in pathological conditions. Furthermore, the presence of substances in caveolae and in their membranes may imply a role in the importing and exporting of key molecules under physiological and pathological conditions. Secretory activity is also suggested by an electron micrograph of rat heart muscle.

Numerical simulation of pulsating flow in the aortic arch.

In order to investigate the flow profiles in the aorta a numerical three-dimensional model of the aortic arch was created. The velocity fields were simulated by applying an inlet velocity corresponding to the physiological velocity of the pressure wave at the aortic valve. The velocity field distribution was found to be uniform throughout the model during the time of increasing inlet velocities. With decreasing inlet velocities a region of low flow developed in the descending portion of the model leading to recirculating flow at the inner wall. At this region of low flow the variation in velocity with time at the inner wall was approximately twice the variation at the outer wall. As a result of the recirculating flow, the wall shear stresses at the inner wall are low and oscillating, predisposing to the development of atherosclerosis. This model shows that transient fluid flow in the aortic arch can be simulated. Biological studies are needed to prove that this model can be used to predict sites of pathology.

Does urokinase play a role in renal stone formation?

Renal stone formation is a complex multifactorial disease, and it is believed that the initial step in the pathogenesis of urolithiasis must be the precipitation of an organic matrix of mucoproteins followed by precipitation of minerals onto this matrix. An important factor in this process may be the activity and/or concentration of the urinary enzyme, urokinase, which would affect the level of urinary mucoproteins such as uromucoid. In support of this hypothesis, ELISA studies were conducted to investigate the urokinase concentrations in urine obtained from males (22-60 years) with and without renal stones. These results showed a significant decrease in urinary urokinase concentration of renal stone patients which, once again, underlines the possible involvement of urokinase in renal stone formation. Therefore, it seems logical to conclude that urokinase may play an integral role in this multifactorial disease.

The conservation of mitochondrial function by ischaemia--the ischaemia paradox.

The aim of this study was to compare oxidative phosphorylation of rat heart mitochondria, isolated: (i) immediately after excision of the heart; (ii) after 60 min of low flow normoxic perfusion; (iii) after 60 min of high flow normoxic perfusion;: (iv) after 30 min of no-flow ischaemia and (v) after 30 min ischaemia followed by 20 min reperfusion. The results, using a retrogradely perfused rat hart as a model, show that mitochondrial oxidative phosphorylation after 30 min of no-flow ischaemia showed no difference from oxidative phosphorylation measured on mitochondria isolated directly after excision of the heart. However, after 30 min no-flow ischaemia followed by 20 min of reperfusion, oxidative phosphorylation deteriorated in comparison with oxidative phosphorylation after 30 min of ischaemia only. Furthermore, oxidative phosphorylation after 60 min of non-ischaemic high flow perfusion (thus high coronary flow), compared better with oxidative phosphorylation after ischaemia-reperfusion than with oxidative phosphorylation after ischaemia alone.

Acclimatization, preconditioning and heat shock proteins.

During prior acclimatization, mineworkers acquire tolerance against the adverse effects of working in a warm environment. In this article, the possible involvement of heat shock proteins as mediators of acclimatization is proposed. Acclimatization is compared with preconditioning. Preconditioning of isolated cells or organs by prior exposure to a temperature higher than normal or exposure to an ischaemic insult endow tolerance on them when later confronted with a severe ischaemic stress. This tolerance is possibly mediated by heat shock proteins induced by the heating or ischaemic preconditioning episode. Functioning of the induced heat shock proteins may therefore underlie the protective mechanisms in both acclimatization and preconditioning.

The possible role of sodium-potassium adenosine triphosphatase in preterm labour.

An unacceptably high incidence of preterm labour is seen in the black and coloured communities of South Africa. This hypothesis proposes that sodium-potassium adenosine triphosphatase activity plays an important role in preterm labour. The impaired activity of the sodium pump leads to increased cytosolic calcium levels, which may trigger contraction of myometrial smooth-muscle cells, resulting in preterm labour.

The effect of different n-6/n-3 essential fatty acid ratios on calcium balance and bone in rats.

Prostaglandins (PGs) are known to have various effects on bone metabolism. The supplementation of essential fatty acids (EFAs), the precursors of PGs, leads to increased intestinal calcium absorption and calcium balance. It is, however, not known whether increased calcium absorption and calcium balance will enhance the calcium content in bone. Male Sprague-Dawley rats (n = 40) aged 5-12 weeks were supplemented with EFAs. The main dietary EFAs, linoleic acid (LA) and alpha-linolenic acid (ALA) were administered in a ratio of 3:1 as a control group. The conversion of LA to ALA to the PG precursors is slow, with the first step, delta-6-desaturation being rate limiting. Fatty acids beyond this rate-limiting step, gamma-linolenic acid (GLA, n-6) and eicoapentaenioc acid (EPA, n-3), were administered to different groups in the ratios 3:1, 1:1 and 1:3 to explore the impact of different ratios of n-6 and n-3 EFAs. Intestinal calcium absorption (mg/24 h) increased by 41.5% in the 3:1 supplemented group, compared with the control group. The decrease in urinary calcium (mg/24 h) correlated with the increase in n-3 level. The calcium balance (mg/24 h) and bone calcium (mg/g bone ash) increased significantly in the 3:1 (41.5% and 24.7%) group, compared with the control. The increase in bone calcium might be attributed to an EFA-induced increase in circulating PGs. An increased synthesis of PGs acting on target bone cells, as well as changes in membrane fluidity, may underlie these observations.

A possible role for inositol 1,4,5-trisphosphate (IP3) in malignant hyperthermia.

In Malignant Hyperthermia an increased open probability of the ryanodine Ca(2+)-channels in the SR-membrane primes a higher cytosolic Ca(2+)-concentration. This in turn accelerates ATP-hydrolysis culminating in a gradual decrease in ATP-levels. At low ATP-levels, IP3-synthesis is being stimulated and the ability of IP3 to release Ca2+ is enhanced. This results through the opening of IP3-dependent channels. Ca(2+)-release through the ryanodine channels and the IP3-dependent channels summate to increase the cytosol Ca(2+)-levels to such a high value that ATP is hydrolysed below a critical value causing rigor (contracture) of skeletal muscle. Since Ca(2+)-uptake by the SR is ATP-dependent, Ca(2+)-uptake will eventually also slow down and so contributes to the rise in cytosol Ca(2+)-concentration.

[Catecholamines--therapeutic and adverse effects on the heart]. / Katesjolamiene--beskermers en beskadigers van die hart.

Catecholamine activation enhances the inotropy of the heart by increasing the sarcolemmal influx of Ca2+. This increased influx is counteracted by an increased sarcolemmal efflux and sarcoplasmic reticulum uptake of Ca2+. Thus the intracellular milieu is protected against a gradual rise in Ca2+ concentration. However, under conditions of continuous, excessive catecholamine release, the heart's potential to remove Ca2+ from the cytosol might become exhausted. This might be caused by a Ca2+-dependent exhaustion of high-energy phosphates. As a result of this, Ca2+ overload of the myocytes and eventually a decrease in the pump function of the heart might occur. This paradox has implications for the clinical management of ischaemic heart disease.

Inhibition of contractility during the early phase of total ischaemia in the working heart. Recovery during reperfusion.

Total global ischaemia of the normothermic working rat heart caused an initial positive inotropic response characterized by vigorous contractions. After +/- 15 s this response reached a peak whereafter the isotonic contraction amplitude started to decline. After +/- 3.5 min the heart ceased to beat. The low level of high energy phosphates (HEP), determined 3 min after the onset of ischaemia, indicated that these phases of contractility during ischaemia might play a significant role in depleting HEP. This was substantiated by the observation that inhibition of the contractions during ischaemia by low calcium or high potassium solutions resulted in conservation of myocardial adenosine triphosphate (ATP) and creatine phosphate (CP) stores. It also resulted in the prevention of contracture development during ischaemia and improved mechanical recovery during reperfusion. It was therefore concluded that inhibition of contractility immediately after the onset of total global ischaemia of the normothermic working rat heart is of prime importance in mechanical recovery during reperfusion.