In situ measurements of enzyme activities in the brain

Summary The present review focusses on enzymes involved in the metabolism of amino acid neurotransmitters and the microphotometric determinations of their activities in various layers of the rat hippocampus. The enzymes are NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), and GABA transaminase (GABAT), all of which are localized in mitochondria. GDH seems to be restricted to astrocytes, whereas NAD-ICDH and GABAT are localized in neurons as well as in astrocytes. NAD-ICDH is an important enzyme of the tricarboxylic acid cycle and may deliver -ketoglutarate for the formation of glutamate and GABA, which serve as neurotransmitters in the hippocampus. GDH catalyses the interconversion of -ketoglutarate and glutamate, whereas GABAT is the important GABA-degrading enzyme and requires -ketoglutarate for its activity. While differing in their cellular distribution and activity levels, NAD-ICDH, GDH and GABAT are significantly correlated in their hippocampal distribution. Furthermore, developmental and pharmacohistochemical studies suggest that the distribution and activity of astrocytic GDH is correlated with amino-acidergic neurotransmission in the hippocampus. The data reported give further evidence for a metabolic relationship between neurons and astrocytes in the turnover and metabolism of glutamate and GABA.     

Age-related quantitative changes in enzyme activities of rat brain

The patterns of brain enzymes linked to energy metabolism have been determined in rats aged between 3 and 21 months and compared to those of the developing brain as an estimate of the senescent energy capacity of this organ. During aging, pyruvate kinase increases, pointing towards an enhancement of the glucose-dependence of this organ. However, NAD-isocitrate dehydrogenase declines, suggesting a reduction of Krebs cycle activity in the aged rat brain. An increase in cytoplasmic NAD-malate dehydrogenase found during aging could provide an alternative mechanism of NAD recovery.     

Quantification of enzyme activities in brain sections by microphotometry

1. Catalytic enzyme histochemistry offers the possibility to demonstrate enzyme activities quantitatively (microphotometry) in brain sections of those sites where they are localized. 2. A prerequisite for quantification are appropriate histochemical procedures for the demonstration of enzymes, which are shortly discussed. 3. For the microphotometric determination of enzymes in brain sections the scanning microphotometry is at present the technique of choice. 4. This is described in the example of an image plane scanning system. 5. Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. kinetic and end-point measurements. 6. Methods for the microphotometric determination of certain important oxido-reductases and further enzymes are presented. 7. It is concluded that quantitative catalytic enzyme histochemistry could be a source of results complementary to those provided by conventional biochemistry.     

In situ assembly of enzyme inhibitors using extended tethering

Cysteine aspartyl protease-3 (caspase-3) is a mediator of apoptosis and a therapeutic target for a wide range of diseases. Using a dynamic combinatorial technology, 'extended tethering', we identified unique nonpeptidic inhibitors for this enzyme. Extended tethering allowed the identification of ligands that bind to discrete regions of caspase-3 and also helped direct the assembly of these ligands into small-molecule inhibitors. We first designed a small-molecule 'extender' that irreversibly alkylates the cysteine residue of caspase-3 and also contains a thiol group. The modified protein was then screened against a library of disulfide-containing small-molecule fragments. Mass-spectrometry was used to identify ligands that bind noncovalently to the protein and that also form a disulfide linkage with the extender. Linking the selected fragments with binding elements from the extenders generates reversible, tight-binding molecules that are druglike and distinct from known inhibitors. One molecule derived from this approach inhibited apoptosis in cells.     

Histochemical localization of capillary enzyme activities in brain smears.

Since the regulation of the vascular permeability in the CNS is dependent partly on the enzymes associated with the wall of brain capillaries, the histochemical demonstration of these enzymes may furnish further data on the function of the blood brain barrier (BBB), a new methodological approach, using brain smears was developed for the histochemical demonstration of several enzymes participating in the function of the BBB. The method presented renders possible also the subsequent demonstration of monoamines and the activity of different enzymes in the same tissue preparation. The usefulness of this simple technique in the study of brain capillary functions is discussed.     

In situ measurements of carbon dioxide gradients in a soil-plant-atmosphere system

Summary In order to more fully understand carbon dioxide dynamics in a soil-plant-atmosphere system, an in situ sampling technique has been developed to measure carbon dioxide concentration within the soil profile as well as in the atmosphere. Gas samples are automatically pumped in sequence from six porous collectors within the soil profile and five aboveground inlets through an infrared gas analyzer. Field measurements in a first year field, indicated that carbon dioxide concentrations reached a maximum value (1800 ppm) in the deepest soil sampling site (-180 cm). Temporal and spatial variations of carbon dioxide concentration were related to the development of root and vegetation structure as well as the position of the groundwater table.Supported, in part, by a grant from the Graduate Research Board. We acknowledge, with thanks, the competent assistance of Frank W. Schwartz.     

In situ measurements of viral particles diffusion inside mucoid biofilms

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections.     

In situ methods for assessment of microorganisms and their activities

Recent technical developments in the field of molecular biology and microsensors are beginning to enable microbiologists to study the abundance, localization and activity of microorganisms in situ. The various new methods on their own bear high potential but it is the combination of studies on structure and function of microbial communities that will yield the most detailed insights in the way microorganisms operate in nature.     

In situ measurements of net primary production in a Colorado mountain stream

Six stations were established on a Colorado mountain stream, and net primary productivity was measured in situ during all seasons. For 24-hour periods the dissolved oxygen and temperature of the water were electronically monitored over an undisturbed 1 x 1 m section of rubble bottom enclosed by a large plastic dome tightly fitted to the substrate. A submerged pump maintained a current within the dome, and the whole apparatus was submerged below the stream level. The bottom community net metabolism varied between heterotrophy and autotrophy with no correlations with altitude, season, light, water chemistry, and temperature. Readings were all very low and ranged from -27.38 to 35.59 grams of carbon fixed per square meter per year. There were no correlations between biomass of the bottom fauna and net community productivity.Contribution No. 71, University of Colorado Limnology Laboratory     

Sex-dependent antioxidant enzyme activities and lipid peroxidation in ageing mouse brain

We investigated whether oxidant status and antioxidant enzyme activities during ageing of mouse brain are regulated in sex-dependent manner. In the homogenate from the brain of 1, 4, 10 and 18 months old male and female CBA mice, lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx) were determined. LPO was age- and sex-related, favoring males over females throughout the lifespan with the peak in both sexes at 10 months of age. Throughout ageing, no difference in tSOD activity between male and female brains was observed, except in immature 1 month old mice. Gender-related difference in Gpx activity was observed, with higher level in females comparing to males, reaching statistical significance in senescent (18 months old) animals. CAT activity was drastically changed with ageing in both the male and female brain. We found different age associated trends in CAT activity in males and females: decreased with age in males and increased with age in females. Taken together, the present findings indicate that brains of female mice have lower oxidant and higher antioxidant capacity mostly related to CAT and to a lesser extent to Gpx activity.     

In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin

Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and in the biosynthesis of bile acids, cholesterol, and ether phospholipids. Even though maintenance of a stable acid-base milieu is essential for proper peroxisomal function, the determination of the peroxisomal pH (pH(p)) remains inconclusive, and little is known about its regulation. To measure the pH of intact peroxisomes in situ, we used the peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver a pH-sensitive mutant of the green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper targeting was verified by colocalization with the peroxisomal marker catalase. Peroxisomes were visualized by imaging fluorescence microscopy, and ratiometric measurements were combined with calibration using ionophores or a null-point method to estimate pH(p). The pH(p) was between 6.9 and 7.1, resembling the cytosolic pH. Manipulation of the cytosolic pH in intact cells or after permeabilization of the plasmalemma with streptolysin O revealed that pH(p) changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H(+) (equivalents). We conclude that peroxisomes do not regulate their pH independently, but instead their large H(+) permeability effectively connects them with the buffer reservoir of the cytoplasm and with the homeostatic mechanisms that control cytosolic pH.     

Effects of acute footshock stress on antioxidant enzyme activities in the adolescent rat brain

In a previous study we demonstrated that acute footshock stress increased glutathione peroxidase activity in the prefrontal cortex and striatum of adult male rats. Adolescents may respond differently to stress as life stressors may be greater than at other ages. The present study examined the effects of the acute footshock stress on superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities and thiobarbituric acid reactive substances (TBARS) levels in adolescent male and female rat brains. We demonstrated that acute footshock stress increased SOD activity in the prefrontal cortex, and increased GPx activity in the hippocampus in female rats. In males, acute footshock stress increased GPx activity in the prefrontal cortex and hippocampus. Footshock stress did not change TBARS levels. These results indicate a strong role of gender in the response of adolescent subjects to various aspects of stress.     

Mass-spectrometric measurements of P450 isoform specific content and corresponding enzyme activities

Mouse cytochrome P450 subfamily 1A, 3A, 1E, 2C, 2D isoenzyme activities and corresponding contents were measured by means of triple quadrupole mass spectrometer in Multiple Reaction Monitoring mode (MRM). The technique was developed and tested on microsomes from control mouse and after induction with phenobarbital or methylcholanthrene. MRM allowed us to measure the content of individual P450 isoforms without using isotopic-labeled peptides or derivatization reagent. The results of modifying the content of certain P450 isoforms correlated with the change of enzymatic activity, defined by marker substrates.     

In situ detection and characterization of DNA polymerase activities in the nucleus of eukaryotic cells

We have developed a cytoenzymological method for localizing DNA polymerase activities in situ and for studying their responses to various chemical agents or environmental conditions. The incubation mixtures and the stimulatory or inhibitory agents added to these media were defined with reference to in vitro biochemical tests used to detect and to characterize DNA polymerases- or - found in eukaryotic cells. This method has already been used to study DNA polymerase activities during cell differentiation or cell senescence. Apart from two exceptions found with lower organisms, the nuclear DNA polymerase activity was always higher under conditions which favoured the in vitro expression of DNA polymerase- rather than DNA polymerase-. — In the various cell types studied, the cellular DNA polymerase activities were almost exclusively found in the nuclei. It is hoped that this methodology will be useful for obtaining more complete biochemical data on the intracellular localization of various DNA polymerases.