Good servant, bad master: sulfide influence on partial nitritation of sewage.

When applying partial nitritation (PN) to anaerobically pre-treated sewage, ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) will be exposed to dissolved sulfide and methane. Both sulfide and methane may inhibit nitrification. To gain knowledge necessary for sustaining PN under these conditions, we exposed an AOB enrichment and a mixed nitrifying culture to dissolved sulfide and methane. In the mixed nitrifying culture, sulfide selectively inhibited NOB activity (KI,AOB1 = 150 mg-S L-1, KI,NOB = 10 mg-S L-1) which shows that sulfide may help establish PN. The AOB enrichment showed similar KI,AOB2 (130 mg-S L-1), but nitritation activity lagged longer than the time necessary to remove sulfide from the liquid. This demonstrates that feeding of sulfide into established PN should be avoided. Methane inhibition of AOB enrichment was assessed in batch assays with 10 mg-CH4 L-1. As compared to control without methane, AOB enrichment activity was identical. Up to 51% of methane was converted to methanol, thus reducing the greenhouse gas emissions.

Whole-body vibration training induces hypertrophy of the human patellar tendon.

Animal studies suggest that regular exposure to whole-body vibration (WBV) induces an anabolic response in bone and tendon. However, the effects of this type of intervention on human tendon properties and its influence on the muscle-tendon unit function have never been investigated. The aim of this study was to investigate the effect of WBV training on the patellar tendon mechanical, material and morphological properties, the quadriceps muscle architecture and the knee extension torque-angle relationship. Fifty-five subjects were randomized into either a vibration, an active control, or an inactive control group. The active control subjects performed isometric squats on a vibration platform without vibration. Muscle and tendon properties were measured using ultrasonography and dynamometry. Vibration training induced an increase in proximal (6.3%) and mean (3.8%) tendon cross-sectional area, without any appreciable change in tendon stiffness and modulus or in muscle architectural parameters. Isometric torque at a knee angle of 90° increased in active controls (6.7%) only and the torque-angle relation remained globally unchanged in all groups. The present protocol did not appreciably alter knee extension torque production or the musculo-tendinous parameters underpinning this function. Nonetheless, this study shows for the first time that WBV elicits tendon hypertrophy in humans.

Immediate effects of whole body vibration on patellar tendon properties and knee extension torque.

PURPOSE: Reports about the immediate effects of whole body vibration (WBV) exposure upon torque production capacity are inconsistent. However, the changes in the torque-angle relationship observed by some authors after WBV may hinder the measurement of torque changes at a given angle. Acute changes in tendon mechanical properties do occur after certain types of exercise but this hypothesis has never been tested after a bout of WBV. The purpose of the present study was to investigate whether tendon compliance is altered immediately after WBV, effectively shifting the optimal angle of peak torque towards longer muscle length. METHODS: Twenty-eight subjects were randomly assigned to either a WBV (n = 14) or a squatting control group (n = 14). Patellar tendon CSA, stiffness and Young's modulus and knee extension torque-angle relationship were measured using ultrasonography and dynamometry 1 day before and directly after the intervention. Tendon CSA was additionally measured 24 h after the intervention to check for possible delayed onset of swelling. RESULTS: The vibration intervention had no effects on patellar tendon CSA, stiffness and Young's modulus or the torque-angle relationship. Peak torque was produced at ~70° knee angle in both groups at pre- and post-test. Additionally, the knee extension torque globally remained unaffected with the exception of a small (-6%) reduction in isometric torque at a joint angle of 60°. CONCLUSION: The present results indicate that a single bout of vibration exposure does not substantially alter patellar tendon properties or the torque-angle relationship of knee extensors.

Alpine Skiing With total knee ArthroPlasty (ASWAP): muscular adaptations.

This study investigated the effectiveness of recreational skiing as an intervention to improve quadriceps muscle architecture, strength, and antagonistic co-activation in patients with unilateral total knee arthroplasty (TKA). Hence, patients with TKA were assigned to either an intervention group (IG) or control group (CG). The IG completed a 12-week guided skiing program whereas the CG was instructed not to change their daily routines for the same period and was not allowed to ski. Before, after the intervention/after an 8-week retention period m. rectus femoris (RF) cross-sectional area (CSA), m. vastus lateralis muscle thickness, fascicle length, and pennation angle were measured with ultrasonography, while isometric (90° knee angle) knee extension, flexion torque and m. biceps femoris co-activation were assessed on an isokinetic dynamometer in 26 patients. There were significant and stable increases in RF CSA for the operated (10%; P < 0.05) and non-operated leg (12%; P < 0.01) after the training period in the IG whereas no changes were observed for the CG (all P > 0.05). There were no significant effects for other parameters (all P > 0.05). Overall, the skiing intervention was successful in increasing muscle mass in TKA older patients.

Alpine Skiing With total knee ArthroPlasty (ASWAP): effect on tendon properties.

The aim of this study was to investigate the effect of alpine skiing on patellar tendon properties in patients with total knee arthroplasty (TKA). Thirty-one adults (70.4 ± 4.7 years) with unilateral TKA were recruited 2.7 ± 0.9 years after surgery and assigned to an intervention (IG) or a control group (CG). The IG underwent a 12-week guided skiing program. Tendon stiffness, Young's modulus, and cross-sectional area (CSA) were measured before and after the intervention. In both groups, mean tendon CSA was 28% (P < 0.001) larger in the operated (OP) than in the non-operated (NOP) leg at baseline, without any difference in other tendon properties. After training, stiffness increased in the IG by 5.8% and 15.8%, respectively, in the OP and NOP legs. Likewise, mean CSA increased in the IG by 2.9% in the OP and 3.8% in the NOP leg, whereas no significant changes were found for the Young's modulus. None of the tendon parameters changed in the CG. Results indicate that patellar tendon structure and/or loading pattern are altered following TKA, but this tissue seems to retain its adaptation capacity. Further, alpine skiing appears to offer a suitable rehabilitation strategy for TKA patients.

Sagopilone (ZK-EPO, ZK 219477) for recurrent glioblastoma. A phase II multicenter trial by the European Organisation for Research and Treatment of Cancer (EORTC) Brain Tumor Group.

BACKGROUND: Sagopilone (ZK 219477), a lipophylic and synthetic analog of epothilone B, that crosses the blood-brain barrier has demonstrated preclinical activity in glioma models. PATIENTS AND METHODS: Patients with first recurrence/progression of glioblastoma were eligible for this early phase II and pharmacokinetic study exploring single-agent sagopilone (16 mg/m(2) over 3 h every 21 days). Primary end point was a composite of either tumor response or being alive and progression free at 6 months. Overall survival, toxicity and safety and pharmacokinetics were secondary end points. RESULTS: Thirty-eight (evaluable 37) patients were included. Treatment was well tolerated, and neuropathy occurred in 46% patients [mild (grade 1) : 32%]. No objective responses were seen. The progression-free survival (PFS) rate at 6 months was 6.7% [95% confidence interval (CI) 1.3-18.7], the median PFS was just over 6 weeks, and the median overall survival was 7.6 months (95% CI 5.3-12.3), with a 1-year survival rate of 31.6% (95% CI 17.7-46.4). Maximum plasma concentrations were reached at the end of the 3-h infusion, with rapid declines within 30 min after termination. CONCLUSIONS: No evidence of relevant clinical antitumor activity against recurrent glioblastoma could be detected. Sagopilone was well tolerated, and moderate-to-severe peripheral neuropathy was observed in despite prolonged administration.

Phase I study of the novel, fully synthetic epothilone sagopilone (ZK-EPO) in patients with solid tumors.

BACKGROUND: Sagopilone (ZK-EPO) is a fully synthetic microtubule-stabilizing agent that has demonstrated high antitumor activity in preclinical models. This first-in-human phase I study aimed to determine the maximum tolerated dose (MTD) and dose-limiting toxic effects (DLTs) of 3-weekly sagopilone treatment. PATIENTS AND METHODS: A total of 52 patients with advanced solid tumors received a 30-min infusion of escalating doses of sagopilone (0.6-29.4 mg/m(2)) every 3 weeks. Nine additional patients were recruited to a 3-h infusion arm (16.53- or 22.0-mg/m(2) dose) to assess the incidence of neuropathy with prolonged infusion. RESULTS: The MTD was established as 22.0 mg/m(2). DLTs comprised peripheral sensory neuropathy (PNP), infection, hyponatremia, diarrhea, and central ataxia. PNP was the most common grade 3 event, with a similar incidence in the 30-min and 3-h arms. Hematologic adverse events were rare and of low intensity. One confirmed partial response (PR) and one unconfirmed PR were reported in the 30-min arm, and a further unconfirmed PR was observed in the 3-h arm. Eleven patients achieved disease stabilization. Sagopilone showed high levels of tissue binding and no obvious serum accumulation in both arms. CONCLUSIONS: These data demonstrate that sagopilone therapy is feasible and well tolerated. The recommended dose for phase II studies is 16.53 mg/m(2), once every 3 weeks.

Weekly administration of sagopilone (ZK-EPO), a fully synthetic epothilone, in patients with refractory solid tumours: results of a phase I trial.

BACKGROUND: Epothilones are a novel class of microtubule-stabilising agents, and sagopilone is a fully synthetic epothilone that has shown marked in vivo and in vitro preclinical activity. METHODS: This phase I, open-label study investigated the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of weekly sagopilone. Twenty-three patients with malignancy resistant or refractory to standard treatment were enrolled into this study evaluating sagopilone doses from 0.6 to 7.0 mg m(-2). RESULTS: The incidence of drug-related haematological adverse events (AEs) was low, with two grade 3 events observed. Nonhaematological AEs were generally mild and reversible; increased gamma-GT was the only grade 4 event and grade 3 events comprised peripheral neuropathy (n=2), diarrhoea (n=1) and fatigue (n=1). Two grade 3 events were DLTs (diarrhoea and peripheral neuropathy at 7.0 mg m(-2)). The MTD of weekly sagopilone was therefore established as 5.3 mg m(-2). Stable disease was the best overall response (n=3). Microtubule bundle formation in peripheral blood mononuclear cells increased post-treatment, peaking after 1 h. Sagopilone disposition was similar across treatment courses and showed rapidly decreasing serum concentrations after infusion end and a long terminal disposition phase with no obvious accumulation in the serum, probably reflecting a fast uptake into tissues followed by a slow release. CONCLUSION: Weekly administration of sagopilone could represent an alternative to the 3-weekly administration currently evaluated in phase II trials.

Arginine metabolism and the synthesis of nitric oxide in the nervous system.

The biochemistry and physiology of L-arginine have to be reconsidered in the light of the recent discovery that the amino acid is the only substrate of all isoforms of nitric oxide synthase (NOS). Generation of nitric oxide, NO, a versatile molecule in signaling processes and unspecific immune defense, is intertwined with synthesis, catabolism and transport of arginine which thus ultimately participates in the regulation of a fine-tuned balance between normal and pathophysiological consequences of NO production. The complex composition of the brain at the cellular level is reflected in a complex differential distribution of the enzymes of arginine metabolism. Argininosuccinate synthetase (ASS) and argininosuccinate lyase which together can recycle the NOS coproduct L-citrulline to L-arginine are expressed constitutively in neurons, but hardly colocalize with each other or with NOS in the same neuron. Therefore, trafficking of citrulline and arginine between neurons necessitates transport capacities in these cells which are fulfilled by well-described carriers for cationic and neutral amino acids. The mechanism of intercellular exchange of argininosuccinate, a prerequisite also for its proposed function as a neuromodulator, remains to be elucidated. In cultured astrocytes transcription and protein expression of arginine transport system y(+) and of ASS are upregulated concomittantly with immunostimulant-mediated induction of NOS-2. In vivo ASS-immunoreactivity was found in microglial cells in a rat model of brain inflammation and in neurons and glial cells in the brains of Alzheimer patients. Any attempt to estimate the contributions of arginine transport and synthesis to substrate supply for NOS has to consider competition for arginine between NOS and arginase, the latter enzyme being expressed as mitochondrial isoform II in nervous tissue. Generation of NOS inhibitors agmatine and methylarginines is documented for the nervous system. Suboptimal supply of NOS with arginine leads to production of detrimental peroxynitrite which may result in neuronal cell death. Data have been gathered recently which point to a particular role of astrocytes in neural arginine metabolism. Arginine appears to be accumulated in astroglial cells and can be released after stimulation with a variety of signals. It is proposed that an intercellular citrulline-NO cycle is operating in brain with astrocytes storing arginine for the benefit of neighbouring cells in need of the amino acid for a proper synthesis of NO.

Effect of astroglial cell swelling on pH of acidic intracellular compartments.

A variety of pathological conditions lead to swelling of astrocytes, which in turn stimulates ion release by activation of ion channels at the plasma membrane. In the present study, acridine orange and fluorescein isothiocyanate coupled to dextran (FITC-dextran) have been used to examine the effect of cell swelling on pH in acidic compartments of cultured astroglial cells. Both NH4Cl (2 mM) and chloroquine (10 microM), known to alkalinize acidic cellular compartments, led to the expected increase in acridine orange fluorescence intensity. Similar, albeit smaller, effects were elicited by a reduction of extracellular osmolarity (-80 mOsm) and treatment of the cells with glutamate (l mM), manoeuvres which enhanced cell volume. Determination of changes in the FITC-dextran fluorescence ratio (485/440 nm) allowed quantification of the pH changes in lysosomal compartments. Treatment with NH4Cl, reduced extracellular osmolarity and glutamate increased lysosomal pH by 0.65 +/- 0.07, 0.85 +/- 0.14 and 0.25 +/- 0.07, respectively. Measurement of cytosolic pH using 2',7',-bis-(2-carboxyethyl)-5- (and -6) carboxyfluorescein (BCECF) demonstrated a pronounced acidification following cell swelling, observed with both reduced extracellular osmolarity (by 0.23 +/- 0.05 pH units) and 1 mM glutamate (by 0.26 +/- 0.02 pH units). In conclusion, pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astrocyte function.

Photoperiod and thermoregulation in vertebrates: body temperature rhythms and thermogenic acclimation.

Evidence has recently begun to accumulate that photoperiodic responses of mammals and birds may affect the control of energy balance and thermoregulation. Exposure to short photoperiod can lower the set point for body temperature regulation in birds and mammals, as well as the voluntarily selected body temperature in ectothermic lizards. This decrease is accompanied by a reorganization of circadian or ultradian rhythms of body temperature, particularly an increase in periods spent at rest with minimum body temperatures. Short photoperiod is also used as an environmental cue for induction of seasonal torpor or facilitation of hibernation. During winter, cold tolerance of small mammals is improved by an increase of nonshivering thermogenesis in brown fat. Thermogenic capacity of brown fat (respiratory enzymes, mitochondria, uncoupling protein) is enhanced in response to short photoperiod. This response is mediated via an increase in the activity of sympathetic innervation in brown fat. Moreover, an exposure to short photoperiod prior to low temperatures may act in preparing brown fat for facilitated thermogenesis during acclimation to cold. This shows that photoperiodic control not only affects energy balance indirectly via the control of reproduction or body mass, but may directly interact with central control of thermoregulation and may influence the process of acclimatization.

Neuronal and glial coexpression of argininosuccinate synthetase and inducible nitric oxide synthase in Alzheimer disease.

The enzyme argininosuccinate synthetase (ASS) is the rate limiting enzyme in the metabolic pathway leading from L-citrulline to L-arginine, the physiological substrate of all isoforms of nitric oxide synthases (NOS). ASS and inducible NOS (iNOS) expression in neurons and glia was investigated by immunohistochemistry in brains of Alzheimer disease (AD) patients and nondemented, age-matched controls. In 3 areas examined (hippocampus, frontal, and entorhinal cortex), a marked increase in neuronal ASS and iNOS expression was observed in AD brains. GFAP-positive astrocytes expressing ASS were not increased in AD brains versus controls, whereas the number of iNOS expressing GFAP-positive astrocytes was significantly higher in AD brains. Density measurements revealed that ASS expression levels were significantly higher in glial cells of AD brains. Colocalization of ASS and iNOS immunoreactivity was detectable in neurons and glia. Occasionally, both ASS-and iNOS expression was detectable in CD 68-positive activated microglia cells in close proximity to senile plaques. These results suggest that neurons and astrocytes express ASS in human brain constitutively, whereas neuronal and glial ASS expression increases parallel to iNOS expression in AD. Because an adequate supply of L-arginine is indispensable for prolonged NO generation, coinduction of ASS enables cells to sustain NO generation during AD by replenishing necessary supply of L-arginine.

Induction of argininosuccinate synthetase in rat brain glial cells after striatal microinjection of immunostimulants.

The enzyme argininosuccinate synthetase (ASS) initiates the metabolic pathway leading from L-citrulline to L-arginine, the only physiological substrate of all isoforms of nitric oxide synthases. The presence of ASS in glial cells in vivo was investigated by immunohistochemical methods in a model of rat brain inflammation. Phosphate-buffered saline or a mixture of bacterial lipopolysaccharide and interferon-gamma was injected into the left striatum, and animals were killed 24 hours later. Ipsilateral and contralateral sides of brain sections were incubated with an antiserum against ASS or antibodies against cell-specific markers. In the three areas examined, striatum, corpus callosum, and cortex, a strong induction of ASS immunoreactivity was observed in glial cells after injection of immunostimulants. A detailed quantitative analysis of double-stained sections revealed that ASS was almost exclusively expressed in reactive, ED1-positive microglial cells/brain macrophages in immunostimulant- or sham-injected ipsilateral sides of the sections. Furthermore, ASS/ED1 costaining was observed in perivascular cells. Colocalization of ASS with astroglial marker glial fibrillary acidic protein was given only occasionally after immunostimulation. ASS-positive neurons were detected in control and experimental animals; staining intensity was comparable in both cases. The results suggest that neurons express ASS constitutively, whereas the enzyme is induced in glial cells in response to proinflammatory stimuli. This finding is the first demonstration of an induction of a pathway auxiliary to generation of nitric oxide in brain in response to immunostimulants and provides new insight into neural arginine metabolism.

New vitamin D receptor agonists with decreased metabolic stability.

The aim of the study was the development of vitamin D receptor agonists with decreased metabolic stability for the topical treatment of psoriasis and related hyperproliferative skin diseases. Calcitriol analogues 1, 2, 3, all of which contain modifications in the side chain, were synthesized. The obtained analogues were full agonists when the induction of CD14 expression in HL-60 cells, the induction of 5-lipoxygenase activity in Mono Mac 6 cells, and the inhibition of phytohemagglutinin (PHA)-stimulated lymphocyte proliferation were studied. The EC(50) value of the most active compound 1 was 1.2 nM in the CD14 assay and 1 nM in the 5-lipoxygenase assay, whereas calcitriol gave EC(50) values in these assays of 3.7 and 9 nM, respectively. In the lymphocyte proliferation assay, compound 1 and calcitriol had IC(50) values of 0.3 and 2.8 nM, respectively. All three compounds had receptor binding affinities similar to that of calcitriol. The compounds showed a decreased metabolic stability in rat liver homogenates and had a 50-fold lower affinity for the vitamin D-binding protein than calcitriol, which suggests that calcitriol analogues are metabolized more rapidly after systemic uptake or application. When injected into rats, the analogues displayed an approximately 100-fold lower hypercalcemic effect than calcitriol. In summary, our study presents three new and potent vitamin D receptor agonists with interesting profiles for development as antipsoriatic drugs.

Metabolism of a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group.

We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo.

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)-positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.

Acquisition of blood--tissue barrier--supporting features by hepatic stellate cells and astrocytes of myofibroblastic phenotype. Inverse dynamics of metallothionein and glial fibrillary acidic protein expression.

A number of similarities between astrocytes and hepatic stellate cells (HSC) rose the question whether or not the protective barrier features of blood-tissue interface may be provided by HSC as well. To test this hypothesis, we investigated the presence of metallothionein (MT), a functional marker of blood--brain barrier, in HSC in situ and in cell culture and compared the results with those obtained with astrocytes. The dynamics of MT expression in cultured astrocytes and HSC was investigated by simultaneous labelling of the cells with a monoclonal antibody (MAb MT) against a lysine-containing epitope of the cadmium-induced monomer of MT-I from rat liver and antiserum against glial fibrillary acidic protein (GFAP). Cell activation was estimated by the presence of smooth muscle alpha-actin (SMAA). In immunoblotting, MAb MT recognized monomeric MT protein and proteins in the 30-kDa range; both bands were pronounced in brain and barely visible in liver homogenates. In situ, MAb MT reacted with very few perivascular cells situated in the parenchyma of the liver. Double immunolabelling of brain slices with MAb MT and antiserum against GFAP showed large areas of brain containing cells expressing both MT and GFAP. However, there were also regions in the brain where the cells produced solely GFAP or MT. In liver cell culture, MT was absent from HSC and hepatocytes in early periods of cultivation, during which the cells maintained their original features; however, MT was expressed strongly in HSC during their activation under prolonged culture conditions. Inversely, in astrocytes MT was expressed during early culturing and disappeared from the cells together with SMAA in late culture when GFAP was upregulated. These results suggest that the acquisition of myofibroblastic features by perivascular cells empowers them to establish a protective blood-tissue permeability barrier. In addition, this study shows that, at least in cell culture, an enrichment of perivascular cells in GFAP results in the disappearance of protective functions.

Replacement of glucose by sorbitol in growth medium causes selection of astroglial cells from heterogeneous primary cultures derived from newborn mouse brain.

Primary cultures derived from the brains of newborn mice are quantitatively dominated by astroglial cells, but contain also oligodendroglial, phagocytic and ependymal cells. When confluent cultures are fed with glucose-free growth medium containing 25 mM sorbitol for 14 days, oligodendroglial, phagocytic and ependymal cells are eliminated from the culture, as judged by morphological and immunocytochemical criteria. The remaining cells stain positively for vimentin and glial fibrillary acidic protein and, therefore, can be considered as astroglial cells. Inoculation of freshly dissociated mouse brain cells in the absence of glucose in a sorbitol-containing medium is not possible; however, feeding of the cultures from day 2 on with sorbitol instead of glucose results in a pure astroglial culture at confluency. Therefore glucose-free growth medium supplemented with sorbitol can be considered a selective medium for astroglial cells in primary mouse glial cultures.

Malic enzyme isoforms in astrocytes: comparative study on activities in rat brain tissue and astroglia-rich primary cultures.

Anion exchange chromatography on diethylaminoethyl cellulose was optimized to separate the cytosolic and mitochondrial isoforms of malic enzyme from rat brain. Extracts of adult rat brain and of astroglia-rich primary cultures derived from the brains of newborn rats were analyzed for their content of the two isozymes. In the case of brain tissue 45% of malic enzyme activity was due to the cytosolic isoform. In contrast, in extracts from astroglia-rich primary cultures more than 95% of the total activity was associated with the cytosolic isozyme. From these data it is concluded that the cytosolic rather than the mitochondrial isoform of malic enzyme has prominent functions in astroglial metabolism.

Uptake of L-lactate by cultured rat brain neurons.

The uptake of L-lactate was investigated in neuronal primary cultures derived from embryonic rat brain with a radioactive tracer method. After preincubation of the cells in glucose-free buffer for 30 min, uptake increased with time for at least 10 min. A saturable component of uptake was found with half-maximal uptake at 10 mM lactate. This saturable component was abolished in the presence of 10 mM alpha-cyano-4-hydroxcinnamic acid. In addition, a non-saturable component dominated the uptake at high concentrations of lactate. Uptake was accelerated with decreasing pH, and was inhibited considerably by pyruvate. It is concluded that neurons are endowed with a lactate transport system which resembles in its properties the monocarboxylate carrier of peripheral tissues.