Prenatal stress reduces postnatal neurogenesis in rats selectively bred for high, but not low, anxiety: possible key role of placental 11beta-hydroxysteroid dehydrogenase type 2.

Prenatal stress (PS) produces persistent abnormalities in anxiety-related behaviors, stress responsivity, susceptibility to psychopathology and hippocampal changes in adult offspring. The hippocampus shows a remarkable degree of structural plasticity, notably in response to stress and glucocorticoids. We hypothesized that PS would differentially affect hippocampal neurogenesis in rats selectively bred for genetic differences in anxiety-related behaviors and stress responsivity. Pregnant dams of high anxiety-related behavior (HAB) and low anxiety-related behavior (LAB) strains were stressed between days 5 and 20 of pregnancy. The survival of newly generated hippocampal cells was found to be significantly lower in 43-day-old HAB than in LAB male offspring of unstressed pregnancies. PS further reduced newly generated cell numbers only in HAB rats, and this was paralleled by a reduction in doublecortin-positive cell numbers, indicative of reduced neurogenesis. As maternal plasma corticosterone levels during PS were similar in both strains, we examined placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which catalyses rapid inactivation of maternal corticosterone to inert 11-dehydrocorticosterone and thus serves as a physiological 'barrier' to maternal glucocorticoids. PS significantly increased placental 11beta-HSD2 activity in LAB, but not HAB, rats. We conclude that PS differentially affects the number of surviving newly generated cells and neurogenesis in HAB and LAB rats. The high sensitivity of hippocampal neurogenesis to PS in HAB rats is paralleled by a failure to increase placental 11beta-HSD2 activity after stress rather than by different maternal corticosterone responses. Hence, stress-induced placental 11beta-HSD2 expression may be critical in protecting the fetal brain from maternal stress-induced effects on adult neurogenesis.

[Evaluation of potential risks of abrasive water jet osteotomy in-vivo]. / Evaluation verfahrensspezifischer Risikopotentiale der Wasserabrasivstrahl--Osteotomie in-vivo.

Since the 80's the water jet scalpel is an established tool in some surgical fields. It is used in particular in visceral surgery for preparation of parenchymatous organs. By the addition of biocompatible abrasives, this technique is able to effectively machine hard biological tissues. Free defined cutting geometries can be realised in a non contact process. Therewith this method has crucial advantages compared to conventional osteotomy techniques and gives new impulses to the development in endoprosthetics and correction osteotomies of hollow bones. In the presented work the new developed abrasive water injection jet (AWIJ) was used the first time for in-vivo osteotomies. Aim of this study was the detection of potential thrombembolic effects and wash in effects of the cutting fluid. Hollow bones of the fore and hind leg of 20 house pigs were treated with the new cutting technique. Intraoperative documentation of relevant vital parameters was performed by a multi monitoring system. Thrombembolic effects during the osteotomy were detected by transthoracic Doppler ultrasonography and transesophagale echocardiography. The hollow bones were prepared in consideration of the vascularisation's protection especially in respect to the venous flow. Thrombembolic effects with temporary haemodynamic respectively respiratory consequences could be detected exclusively by using the so called "3-component jet", which consists of 90 vol % of air. The usage of an abrasive suspension enables the airfree dosing of dry soluable abrasives. Thrombembolic effects could not be monitored in this case. Intramedullary fluid in-wash effects as well as resulting electrolytic disorders could not be proven. For abrasive waterjet osteotomies with 3 component jet a relevant risk of thrombembolic effects could be shown. This knowledge has also to be considered for abdominal and neurosurgical applications in the future. Due to the usage of an abrasive suspension this risk can fully be avoided.

Differential effects of periodic maternal separation on adult stress coping in a rat model of extremes in trait anxiety.

We studied interactions of genetic and environmental factors shaping adult emotionality and stress coping, and tested the hypothesis that repeated periodic maternal deprivation (PMD) exerts differential effects on adult behavioral and neuroendocrine stress responsiveness in dependence on the genetic predisposition to either hyper- or hypo-anxiety. Exposure of male Wistar rats bidirectionally bred for either high (HAB) or low (LAB) anxiety-related behavior to PMD between postnatal days 2 and 15 resulted in a behavioral approximation of the selected lines. This was reflected by test-dependent signs of reduced anxiety-related behavior in adult HAB rats and of enhanced levels of anxiety in LAB rats compared with their corresponding unstressed controls. In addition to behavioral parameters, differential effects of PMD were also seen with respect to the responsiveness of the hypothalamo-pituitary-adrenocortical axis to acute stressor exposure (novel environment) in adulthood. The corticotrophin (ACTH) and corticosterone hyper-responses seen in control rats of the HAB line compared with those of the LAB line became attenuated in PMD-HAB rats, whereas PMD did not significantly alter neuroendocrine responses in LAB rats. Thus, as a result of PMD, both ACTH and corticosterone responses became indistinguishable between HAB and LAB rats. Although HAB dams spent more time on the nest with the litter compared with LAB dams during the first 5 days postpartum, licking and grooming behavior did not differ between the lines prior to separation, and was found to be increased to the same extent in both HAB and LAB dams during the first hour immediately after reunion with the pups. In contrast to early life stress, exposure of adult HAB and LAB rats to a 10-day unpredictable stress schedule failed to alter their emotional measures. The mitigating effect of PMD on both behavioral and neuroendocrine parameters in rats representing extremes in trait anxiety might reflect an evolutionary benefit as the genetic variability among individuals of a species is sustained while allowing adequate responses to potentially dangerous stimuli in adulthood dependent on early life conditions.

[Temperature measurements during abrasive water jet osteotomy]. / Wärmeentwicklung bei der Wasser-Abrasivstrahl-Osteotomie.

Working on bone is a major aspect of orthopaedic surgery. Despite its well-known appreciable thermal effects on the edges of the bone cut, the oscillating bone saw blade the oscillating saw remains the standard instrument both for cutting long bones and creating a bed for an endoprosthesis. The application of abrasive water jets offers the possibility of achieving an extremely precise curved cut in bone with no accompanying thermal effect. The thermographically measured absolute temperature increase at the cut edges seen with the water jet was 13 K maximum. The small process forces permit the application in automated handling systems.

Release of oxytocin in the hypothalamic paraventricular nucleus, but not central amygdala or lateral septum in lactating residents and virgin intruders during maternal defence.

In lactating rats, the neuroendocrine responses of the oxytocinergic system and the hypothalamo-pituitary-adrenal axis to various kinds of stressors are attenuated. In this study, using intracerebral microdialysis in combination with a highly sensitive radioimmunoassay, we characterised oxytocin (OXT) release within the paraventricular nucleus (PVN), the central amygdala (CeA), and the medio-lateral septum (mS) before, during and after a psycho-social stressor (the maternal defence test) in both the virgin intruder and the lactating resident rat (day 3 of lactation). Within the PVN, local OXT release was found to increase significantly in virgin intruders during exposure to the resident (2.1-fold, P < 0.05), as well as in lactating residents when exposed to the virgin intruder, though to a lesser extent when compared with basal levels (1.7-fold, P < 0.05). In contrast, OXT release remained unchanged within the CeA and the mS of both virgin intruders and lactating residents. Release of OXT under basal conditions was clearly above the detection limit of the radioimmunoassay, and did not differ between lactating and virgin rats in any of the brain regions studied. Our study also demonstrates that recent surgery or ongoing intracerebral microdialysis does not affect the behavioural performance of the intruders or residents when comparing dialysed and non-dialysed rats. The results indicate that exposure to the maternal defence test is a relevant stressor for the brain OXT system which becomes activated in both intruder and resident rats, although to varying degrees depending upon their reproductive status and in a region-dependent manner. The behavioural and/or neuroendocrine functions of intra-PVN released OXT during this psycho-social challenge remain to be clarified.

Maternal defence as an emotional stressor in female rats: correlation of neuroendocrine and behavioural parameters and involvement of brain oxytocin.

In order to study neuroendocrine and behavioural stress responses in female rats post partum we aimed to establish a relevant emotional stressor -- the maternal defence test based on maternal aggression of a lactating resident towards a virgin or lactating intruder approaching the cage. Exposure to maternal defence significantly elevated corticotropin (ACTH) and corticosterone responses of the residents and of virgin or lactating intruders, with an attenuated response in lactating residents and lactating intruders. Exposure to maternal defence increased plasma oxytocin in virgin intruders only. The aggressive behaviour displayed by the residents was directly correlated with the amount of defensive behaviour of the intruder and independent of the intruder's reproductive state. However, the amount of maternal and explorative behaviours displayed by the lactating residents was significantly higher when exposed to a lactating, compared to a virgin, intruder. ACTH responses in lactating residents exposed to virgin intruders were significantly correlated to the amount of offensive (direct correlation) and maternal (inverse correlation) behaviours they displayed. Plasma prolactin concentrations, elevated in lactating compared to virgin rats under basal conditions, were found to be reduced in the lactating residents and intruders in response to exposure to the maternal defence test, whereas it was unchanged in virgin intruders. To test for the involvement of brain oxytocin in neuroendocrine and behavioural responses of the lactating residents an oxytocin receptor antagonist (0.1 microg/5 microL) was infused icv 10 min prior to testing. This treatment increased basal, but not stress-induced, ACTH, corticosterone and oxytocin secretion. Whereas parameters of aggressive behaviour were unchanged, the antagonist reduced signs of maternal behaviour during maternal defence. In summary, the maternal defence test has been characterized as a relevant emotional stressor for female rats which is useful for studying neuroendocrine and emotional responses in females, in particular in the context of reproductive adaptations.

Brain oxytocin inhibits the (re)activity of the hypothalamo-pituitary-adrenal axis in male rats: involvement of hypothalamic and limbic brain regions.

In response to various stressors, oxytocin is released not only into blood, but also within hypothalamic and extrahypothalamic limbic brain regions. Here, we describe the involvement of intracerebrally released oxytocin in the regulation of the activity of the hypothalamo-pituitary-adrenal (HPA) axis by infusion of the oxytocin receptor antagonist (des Gly-NH(2) d(CH(2))(5) [Tyr(Me)(2), Thr(4)] OVT; pH 7.4; Dr. M. Manning, Toledo, OH, USA) either into the lateral cerebral ventricle (icv[0.75 microg/5 microl,]) or via retrodialysis (10 microg/ml, 3.3 microl/min, 15 min) into the hypothalamic paraventricular nuclei (PVN), the medio-lateral septum or the amygdala. Male Wistar rats fitted with a chronic jugular vein catheter and an icv guide cannula or a microdialysis probe targeting the respective brain region 4 days prior to the experiment were blood sampled under basal as well as stressful conditions. Rats were exposed to the elevated platform (emotional stressor) and/or to forced swimming (combined physical and emotional stressor). Blockade of the receptor-mediated action of endogenous oxytocin within the PVN resulted in an enhanced basal secretion of ACTH whereas, in response to forced swimming, ACTH secretion was rather reduced, indicating a tonic inhibitory effect of OXT on basal HPA axis activity, but a potentiating action under conditions of stress. Within the medio-lateral septum, antagonist treatment did not alter basal ACTH secretion, but significantly disinhibited ACTH secretion in response to the elevated platform, but not to forced swimming. Within the amygdala, no significant effects either on basal or stress-induced HPA axis activity could be found. The results indicate a differential involvement of brain oxytocin in the regulation of the HPA axis activity which depends both on the site of intracerebral oxytocin release and the stressor the animals are exposed to.

Regulation of the supply of cytosolic oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts. I. The effect of covalent modification on PEPC activity, pH response, and kinetic properties.

The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by covalent modification is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Extracts for studies on in vivo PEPC phosphorylation were prepared from barley leaf protoplasts by rapid filtration, fractionating the cell within less than 1 s. Measurements of in vitro PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. The relative PEPC phosphorylation state increased upon illumination and decreased upon redarkening under photorespiratory and non-photorespiratory conditions. PEPC activity measured in the presence of malate (3 mM) under photorespiratory conditions showed the same response indicating that a light-induced increase in PEPC activity and decrease in malate sensitivity is caused by an increased phosphorylation level of the PEPC protein. PEPC activity was pH dependent. At the physiological cytosolic pH, activity was suboptimal, but most sensitive towards malate inhibition and glucose 6-phosphate stimulation. The presence of malate increased the sensitivity of PEPC activity towards pH changes. The response of PEPC activity to changing pH was not affected by changes in the activation state of the enzyme. The Km (phosphoenolpyruvate, PEP) is about 1 mM. Upon illumination the Km (PEP) decrease significantly. Vmax was unaffected by the light treatment. The presence of physiological concentrations of glucose 6-phosphate decreased Km (PEP) 5- to 10-fold and increased Vmax by about 35%. The effect of glucose 6-phosphate was strongest (up to 7-fold) at subsaturating PEP concentrations stimulating PEPC activity to nearly maximal rates. The results show that an increase in PEPC phosphorylation state causes an increase in PEPC activity as well as in substrate affinity leading to an increased production of OAA in the light.

Regulation of the supply of oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts. II. Effects of metabolites on PEPC activity at different activation states of the protein.

The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by metabolites is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Measurements on PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. Glycine, serine, pyruvate, acetyl-CoA, glycolate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and ADP had no significant effect on PEPC activity. Malate, aspartate and glutamate were strong inhibitors of PEPC activity decreasing the activity more in light versus darkness. However, at the physiological cytosolic concentration of these metabolites under the respective conditions, inhibition of PEPC activity was about the same with the exception of aspartate which inhibits more under non-photorespiratory than under photorespiratory conditions. 2-Oxoglutarate and glyoxylate decreased PEPC activity by 20 to 40% in the range of its physiological cytosolic concentration. Inhibition by physiological cytosolic concentrations of glutamine was limited. Glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate, dihydroxyacetonphosphate and P(i) stimulated PEPC activity significantly in their physiological cytosolic concentration range. Physiological cytosolic concentrations of glucose 6-phosphate and fructose 6-phosphate activated PEPC activity to about the same extent under all conditions applied, while 3-phosphoglycerate and dihydroxyacetonphosphate stimulating stronger under non-photorespiratory versus photorespiratory conditions. Moreover, dihydroxyacetonphosphate stimulated PEPC activity more in light versus darkness under non-photorespiratory conditions. P(i) activation of PEPC activity decreases in light versus darkness under non-photorespiratory conditions. Stimulation of PEPC activity by citrate in its physiological concentration range is limited. Glucose 1-phosphate and AMP activated PEPC activity only at concentrations higher than their physiological levels in the cytosol. Determinations of PEPC activity in the presence of different malate/glucose 6-phosphate ratios revealed that glucose 6-phosphate totally relieved the inhibitory effect of malate. The regulatory properties of PEPC activity will be discussed in relation to its functions in C3 plants.

Isolation and purification of mitochondrial Mn-superoxide dismutase from the gymnosperm Pinus sylvestris L.

Manganese superoxide dismutase (Mn-SOD; EC 1.15.1.1) was purified from germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition. The purification schedule included (NH4)2SO4 fractionation, anion-exchange and hydrophobic-interaction chromatographies and chromatofocusing. Purified Mn-SOD had an apparent specific activity of 4,130 McCord-Fridovich units (mg protein)-1. The molecular mass of the holoenzyme was estimated to be 91 kDa by size-exclusion chromatography, and a molecular mass of 23 kDa was determined by SDS-PAGE. However, isoelectric focusing demonstrated that the purified enzyme consisted of three similarly migrating isoforms, with isoelectric points of approximately 6.5. NH2-terminal amino acid sequencing of purified Mn-SOD revealed no differences among the three isoforms. The comparison of the first 32 NH2-terminal amino acids with sequences of NH2-terminal amino acids of Mn-SODs from angiosperms reflected the phylogenetic distances between Scots pine, which is a gymnosperm, and angiospermic species. Cell fractionation suggested the mitochondrial localization of Mn-SODs and no evidence for glyoxysomal localization was found. Mn-SOD activity was absent from dry seeds. It was detectable at a considerable level after imbibition for 24 h, and it was again absent from 3-week-old seedlings.

Mitochondrial Contribution to Photosynthetic Metabolism (A Study with Barley (Hordeum vulgare L.) Leaf Protoplasts at Different Light Intensities and CO2 Concentrations).

An oligomycin concentration that specifically inhibits oxidative phosphorylation was added to isolated barley (Hordeum vulgare L.) leaf protoplasts at various irradiances and carbon dioxide concentrations. At saturating as well as low light intensities, photosynthetic oxygen evolution was decreased as a result of the oligomycin treatment, whereas no effect was observed at intermediate light intensities. This was the same for photorespiratory and nonphotorespiratory conditions. These results were confirmed by measurements of fluorescence quenching under the same conditions. Metabolite analysis in the presence of oligomycin revealed a drastic decrease in the mitochondrial and cytosolic ATP/ADP ratios, whereas there was little or no effect on the chloroplastic ratio. Concomitantly, sucrose phosphate synthase activity was reduced. Under high irradiances, this inhibition of sucrose synthesis by oligomycin apparently caused a feedback inhibition on the Calvin cycle and the photosynthetic activity. Under low irradiances, a feedback regulation compensated, indicating that light was more limiting than the activity of regulative enzymes. Thus, the importance of mitochondrial respiratory activity might be different in different metabolic situations. At saturating light, the oxidation of excess photosynthetic redox equivalents is required to sustain a high rate of photosynthesis. At low light, the supply of ATP to the cytosol might be required to support biosynthetic reactions.

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.

V delta 1+ subset of human gamma delta T cells responds to ligands expressed by EBV-infected Burkitt lymphoma cells and transformed B lymphocytes.

Human gamma delta T cells of peripheral blood can be divided in two groups in terms of their TCR as well as their behavior upon in vitro stimulation. The major subset expresses the TCR V-segments V gamma 9 and V delta 2 and proliferates in response to ligands revealed by various microorganisms, and the cell line Daudi in addition. The minor group is less homogenous on the gamma-chain but is almost completely identified by mAb against the V delta 1 segment; there is no ligand known to promote growth of these cells. Here we demonstrate that gamma delta T cells out of this subgroup are strongly stimulated in vitro by cells from several Burkitt's lymphoma cell lines. EBV infection of the Burkitt's lymphoma cell lines enhanced the stimulatory ability towards the T cells. Although EBV infection influenced the expression of a variety of cell surface molecules including ICAM-1 and LFA-3, no correlation to the gamma delta T cell-stimulating capacity became apparent. We conclude that Burkitt's lymphoma cells and transformed B cells express ligands of cellular origin for a hitherto poorly characterized subgroup of human gamma delta T cells.

Opportunist mycobacteria express ligands that stimulate production of human V gamma 9V delta 2 T lymphocytes.

Human gamma delta T cells are known to respond at high frequencies to pathogenic mycobacteria. Here we show that opportunistic strains of mycobacteria share with pathogenic mycobacteria the ability to trigger at high frequencies human V gamma 9V delta 2 T-cell-receptor-positive T lymphocytes. Stimulating ligands were present in part in a low-molecular-weight fraction of lysates from opportunistic mycobacteria, as has been found for pathogenic strains. These results support the view that postnatal exposure to ever-present opportunistic mycobacteria may be a driving force for the numerical expansion of the V gamma 9V delta 2 T-cell subset in adolescence.

A lectin-binding, protease-resistant mycobacterial ligand specifically activates V gamma 9+ human gamma delta T cells.

Bacterial (exogeneous) superantigens have been defined as bifunctional proteinaceous molecules. They bind to class II MHC molecules of presenting cells and engage with particular TCR-V beta gene elements, thereby activating alpha beta T cells in a V beta-oriented fashion. In previous studies we have elucidated that gamma delta T cells exhibit a propensity to vigorously respond toward mycobacterial Ag. Intrigued by this finding we now analyzed whether mycobacteria express a superantigen for a subset of human gamma delta T cells definable by the selective use of TCR-V gene elements. Here we describe that a protease-resistant, low m.w. (1 to 3 kDa) component of mycobacteria selectively activates gamma delta T cells expressing TCR-V gamma 9 gene segments. Contained in mycobacterial lysates it stimulates TCR-V gamma 9-positive gamma delta T cells at a frequency of 1/6. Stimulation is critically dependent on the presence of class II MHC-positive presenting cells, the important structure being HLA-DR molecules. The fine specificity of the V gamma 9 seeking mycobacterial ligand differs from the gamma delta T cell-stimulating structures expressed by Daudi cells. In addition, the mycobacterial, V gamma 9-seeking ligand is bound selectively to lectins such as UEAI, SBA, and DBA. We conclude that mycobacteria contain a component that acts as a superantigen for human gamma delta T cells and we believe it is this property that explains the vigorous participation of gamma delta T cells in mycobacterial infections.

On the Role of Mitochondrial Oxidative Phosphorylation in Photosynthesis Metabolism as Studied by the Effect of Oligomycin on Photosynthesis in Protoplasts and Leaves of Barley (Hordeum vulgare).

Low concentrations of oligomycin, which strongly inhibit mitochondrial oxidative phosphorylation but do not affect chloroplast photophosphorylation, caused an inhibition of photosynthesis by 30 to 40% in barley (Hordeum vulgare L.) leaf protoplasts. This inhibition is reversed and the full rate of photosynthesis is regained when the protoplasts are ruptured so as to leave the chloroplasts intact. Oligomycin fed into barley leaves by the transpiration stream inhibited photosynthesis in these leaves by up to 60%. The measurement of metabolites in protoplast and leaf extracts showed that oligomycin caused a decrease in the ATP/ADP ratio and an increase in the content of glucose- and fructose 6-phosphate. Subcellular analysis of protoplasts revealed that the decrease in ATP/ADP ratio in the cytosol was larger than in the stroma and that the increase in hexose monophosphates was restricted to the cytosol, whereas the stromal hexosemonophosphates decreased upon the addition of oligomycin. Moreover, oligomycin caused an increase in the triosephosphate-3-phosphoglycerate ratio. It is concluded from these results that during photosynthesis of a plant leaf cell mitochondrial oxidative phosphorylation contributes to the ATP supply of the cell and prevents overreduction of the chloroplast redox carriers by oxidizing reductive equivalents generated by photosynthetic electron transport.

Analysis of primary T cell responses to intact and fractionated microbial pathogens.

Freshly isolated human T lymphocytes were tested for their response to mycobacteria, mycobacterial lysates, 2 dimensional (2D) PAGE separated mycobacterial lysates, leishmania and defined leishmanial antigen preparations. While gamma delta T cells proliferated vigorously in the presence of mycobacteria and mycobacteria derived lysates, a significant stimulation from 2 D gel separated lysates was not detected. In addition gamma delta T cells failed to respond towards leishmania or leishmanial components. In the alpha beta T cell compartment some donors, presumably according to their state of immunity against mycobacteria, responded to mycobacteria, mycobacterial lysates and 2 D gel separated mycobacterial lysates. Neither freshly isolated gamma delta T cells nor alpha beta T cells from naive donors did mount a significant immune response against leishmania.

Oxaloacetate and malate transport by plant mitochondria.

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10(-3). From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.