Inhibition of proprotein convertases-1, -7 and furin by diterpines of Andrographis paniculata and their succinoyl esters.

Studies were performed to investigate the prohormone/proprotein convertase (PC)-inhibitory properties of chemical constituents of the medicinally active plant Andrographis paniculata (AP; from the family Acanthaceae), also known as 'King of Bitters'. Among the individual components tested against the clinically important convertases, furin and PC1, neoandrographolide (a C3 O-glucoside derivative of the major constituent andrographolide) exhibited the highest inhibitory action with an IC50 of 53.5 microM against furin. The data further revealed that although andrographolide, the major bitter principle of AP, exhibited a relatively small enzyme inhibition (IC50=1.0 mM and Ki=200 microM against furin), upon succinoylation, its inhibitory action against the above convertases was enhanced significantly with a Ki in the low micromolar range (<30 microM), suggesting that a specific structural modification of the andrographolide skeleton may be exploited to develop a new class of non-peptide inhibitors of PCs. When tested against PC7, these succinoylated derivatives of andrographolide also displayed strong inhibitory action, with Ki values again in the low micromolar range. This potentially interesting observation may be attributed to the reported anti-HIV property of 14-dehydroandrographolide succinic acid monoester (DASM). It is suggested here that DASM, by virtue of this protease inhibitory property, possibly acts by suppressing the proteolytic cleavage of envelope glycoprotein gp160 of HIV, which is known to be PC-mediated, particularly by furin and PC7.

Dietary calcium supplementation and dopamine-beta-hydroxylase in spontaneously hypertensive rats.

Spontaneously hypertensive 4-week-old male rats were fed, before and after the onset of hypertension, with either commercial chow (control) or commercial chow combined with different forms of milk proteins with or without calcium supplementation. After 40 weeks, rats were still hypertensive, and dopamine-beta-hydroxylase enzyme activity measured simultaneously in serum and adrenal was found to be higher than in the controls. The enzyme activity in rats fed diets with milk proteins was increased significantly in both serum and adrenal compared with the control, and such enhancement was significantly higher than that observed in animals fed the commercial diet supplemented with calcium (1.2%), suggesting that dietary calcium intake associated with dietary protein of high digestibility, such as casein, potentiates the endogenous mechanisms regulating the homeostasis of calcium more than calcium supplementation itself. Moreover, the selective and additive effect of diets supplemented with milk proteins and calcium on adrenal enzyme activity clearly suggests a relationship between cardiovascular diseases involving the genesis of hypertension and stress mechanisms through the hypothalamo-pituitary adreno-sympathetic axis.

Nitric oxide synthase in cat brain: cofactors--enzyme-substrate interaction.

Nitric oxide, derived from L-arginine by the enzyme nitric oxide synthase, is an activator of the soluble guanylate cyclase and a cellular messenger. This work demonstrates that, in cat brain, the neuronal constitutive nitric oxide synthase activity is a) NADPH/calcium dependent, b) independent upon exogenous calmodulin in crude brain supernatant, c) significantly enhanced by exogenous FAD and tetrahydrobiopterin (Vmax: 118 instead of 59.4 pmol of citrulline formed .mg of prot.-1 min-1, d) inhibited by calcium chelators and calmodulin antagonist, and e) present in several neuroanatomical structures. Moreover, the Km value for L-arginine was of 11 microM instead of 41 microM in the presence of FAD and tetrahydrobiopterin in the incubation mixture, thus demonstrating that these cofactors are able to stabilize the enzyme-substrate interactions.

Comparative study of biochemical parameters and kinetic properties of dopamine-beta-hydroxylase activity from cat and rat adrenals.

The specific adrenal dopamine-beta-hydroxylase activity measured in cat and rat was: (1) two times higher in cat than in rat; (2) significantly enhanced by ascorbate (up to 5 mM) that acts as a major activator; (3) differently affected by exogenous copper added to the incubation medium, leading to a decrease with large concentrations; and (4) similar, according to the kinetic parameters, thereby demonstrating a greater affinity for ascorbate than for tyramine.

Long-term effect of low-dose combined steroid contraceptives on body iron status.

The present study was aimed to evaluate iron metabolism in active and healthy adult women having taken oral contraceptives (OC) long-term. Mean dietary iron intake in age-matched control and experimental groups was adequate. Serum ferritin used as a marker for body iron stores was marginal in both groups underlying a high prevalence of deficient-iron reserves among subjects. This parameter was not correlated to the iron content of the diet. The serum iron concentration was significantly higher in OC users than control subjects (p less than 0.001). Biochemical results commanded a discussion on the pertinence of evaluating the total dietary iron intake and on the sensitivity of biochemical methods used to assess the iron status.

Comparative effects of carbohydrate restriction vs starvation on biochemical parameters related to neurotransmitters in rat.

Adult rats were submitted to a 4-day starvation period or maintained on a 50% carbohydrate-restricted diet for 8 consecutive days to obtain a body weight loss of 20-30%. Serum dopamine-beta-hydroxylase (DBH) activity and amino acids content were measured as well as brain tryptophan and tyrosine levels. Moreover, brain serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), noradrenaline (NA), and dopamine (DA) contents were assayed in five brain areas. In 4-day starved and 8-day carbohydrate-restricted rats, the serum tyrosine and total tryptophan contents as well as tyrosine to the sum of six neutral amino acids ratios were lowered. Moreover, in these groups, free tryptophan to the sum of six neutral amino acids ratio remained normal and serum DBH activity increased. In the brain, to a decreased tyrosine content observed in 4-day starved and 8-day carbohydrate-restricted rats corresponded a high DA to NA ratio in the hypothalamus, thalamus, and raphe nuclei, thus suggesting a low DA utilization whereas a low DA to NA ratio was found in the neostriatum. On the other hand, brain tryptophan content was decreased in 4-day starved rats and increased in 8-day carbohydrate-restricted rats. In the former group, a high 5-HT to 5-HIAA ratio characteristic of a low 5-HT utilization was found in the hypothalamus and neostriatum whereas in the latter group a significant decrease in this ratio was only observed in the thalamus. These results suggest that the biochemical response to starvation vs carbohydrate restriction can be differentiated on neurochemical and neuroanatomical bases.

Effects of intrahypothalamic injections of GABA, muscimol, pentobarbital, and L-glutamic acid on feed intake of satiated sheep.

Five wethers were surgically prepared with cranial implants to study the role of gabaminergic neural pathways on the hypothalamic control of feeding behaviour in ruminants. In the first experiment, the animals were injected (1 microL) with a physiological Tyrode (0.95%) solution, muscimol (0.5 and 1.0 nmol), GABA (0.5 and 1.0 nmol), and L-glutamic acid (0.5 and 1.0 nmol). Feed intake following injections of muscimol (1.0 nmol) and L-glutamic acid (0.5 and 1.0 nmol) was twice as large as that following the Tyrode solution, at 60-min postinjections. These results, however, were not statistically significant (p = 0.12-0.15). In the second experiment, the animals were injected (1 microL) with saline, muscimol (0.8 nmol), L-glutamic acid (0.8 nmol), and pentobarbital (0.26 mumol). Fifteen minutes after the injections, pentobarbital had induced a significant feeding response when compared with control values (p less than 0.01), whereas the effect of L-glutamic acid was not significant. However, 30 min after the injections, feed intake of sheep having received L-glutamic acid was higher than that obtained with the control injections (p less than 0.01). The response to pentobarbital was stronger than that to either muscimol or L-glutamic acid. Histological analyses of brain tissue indicated that injections were performed in the ventromedial hypothalamus of four sheep and in the dorsomedial hypothalamus of the other. The data indicate that L-glutamic acid stimulates feed intake by acting either as a precursor of GABA or by a direct stimulation of glutaminergic neural pathways involved in the control of feed intake.

Effects of vitamin B12 supplementation on choline acetyltransferase activity in cat brain.

Vitamin B12 was daily supplemented to a semi-purified casein diet and given per os during 17 consecutive days to cats in a dosage of 25 micrograms/kg every morning at the meal time. The specific choline acetyltransferase activity, measured in several structures in cat brain, was significantly increased in the hypothalamus, piriform lobe, hippocampus, pons and pons raphe nuclei and significantly decreased in the medulla raphe nuclei, respectively. These results demonstrate for the first time that vitamin B12 involved as coenzyme in biochemical reactions related to the liberation of methyl synthesis synthesis through the bioavailability of choline, the enzyme substrate of choline acetyltransferase. These results might contribute to a better understanding of some neurochemical mechanisms related to Alzheimer-type dementia.

Dietary protein and carbohydrate effects on blood parameters related to stress in cat.

Adult cats were adapted to hypoglucidic semi-purified diets containing casein or soya as the protein source to study the effects of a 2 hr immobilization period. Body weight of cats fed hypoglucidic diets was significantly decreased. The control casein group showed higher plasma dopamine-beta-hydroxylase activity but lower pyridoxal 5'-phosphate level than control soya group. Cats fed hypoglucidic casein diet, plasma glucose, insulin and pyridoxal 5'-phosphate levels were increased whereas cats fed hypoglucidic soya diet, pyridoxal 5'-phosphate levels were decreased and dopamine-beta-hydroxylase activity increased when data were compared to their respective control groups. A 2 hr immobilization period induced hyperglycemia in all groups whereas cats fed soya diets, plasma insulin level and dopamine-beta-hydroxylase activity were significantly increased and pyridoxal 5'-phosphate content significantly decreased. These results demonstrate that dietary casein and soya protein might be differentiated on a physiological basis and immobilization emphasized the biochemical disturbances observed between the groups thus suggesting a greater resistance to stress in casein groups than in soya groups.

Influence of diet and hypoxia on brain serotonin and catecholamines in rainbow trout (Salmo gairdneri).

1. In the brain of Salmo gairdneri, the content of dopamine (DA), norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) depends upon the location in the brain (hypothalamus, telencephalon or mesencephalon). 2. The origin of feed protein (from animal or vegetal origin) influences the level of the various monoamines studied in different brain structures. 3. Hypoxia (60% oxygen saturation in water) causes modifications of 5-HT and catecholamine (DA, NE) contents in different brain structures, depending upon the diet.

The nutritional status of subjects with anorexia nervosa.

This study characterized the dietary pattern of 25 subjects with anorexia nervosa from a diet history. The patients mean age was 18 +/- 5 years and mean weight loss from preillness weight was 26%. Diets of anorectics were found to be significantly lower in total energy, reflecting a disturbance in nutrient intake when data were compared to a normal population. The percentage of energy derived from macronutrients was higher for protein and lower for concentrated carbohydrates, but total carbohydrates and lipids were found similar to the ones observed in normal subjects. Dietary intake of calcium, iron, thiamin, riboflavin, niacin and ascorbic acid of anorectics were significantly lower than in normal subjects. Such a particular pattern was paralleled by high serum cortisol levels and circadian cortisol cycle, low morning glycemia, high carotenemia, hypokaliemia and hyperalbuminemia. Results are discussed on the basis of the nutritional adequacy of the anorexia nervosa dietary pattern and its relationship with some aspects of the symptomatology of the disease.

Difference in monoamine oxidase activity measured by either liquid ion exchange or ion exchange resin chromatography in rat and cat brain.

Cat and rat brain monoamine oxidase (MAO) activity was measured with a radioisotopic procedure and two extraction methods. Results indicated an underestimation of MAO activity when liquid ion exchange chromatography (LIEC) was used instead of an ion exchange chromatographic method (IEC) to separate the different products of the deaminated tyramine, phenylethylamine, or serotonin. MAO produced aldehydic products which may be found in the incubation medium and may be extracted with the substrate in the chloroform phase by the LIEC method. In cat brain, the resulting underestimation of the MAO activity was prevented by the addition of nicotinamide adenine dinucleotide (10(-3) M) in the incubation medium or by allowing a 2-h period between the end of incubation and the LIEC extraction procedure. In the rat brain, the same result was obtained by the addition of an equimolar mixture of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate in reduced form (NAD-NADPH, 10(-3) M). Using the IEC method, the NAD decreased only the deamination of tyramine and serotonin in rat brain. This study suggests that the use of an IEC method to evaluate MAO activity is more accurate for the estimation of the enzymatic activity.

Cortisol and catecholamines response to venisection by humans with different blood groups.

Hormonal response following a minor physiological stress induced by bleeding in a blood donor clinic was investigated in 134 humans with different blood groups. After venisection, serum cortisol concentration (mean +/- SD) was found highest in blood group A donors (455 +/- 217 nmol/L), followed by group B (364 +/- 206), AB (325 +/- 154) and O (297 +/- 110). In 14 subjects of blood group A compared to an equal number of blood group B subjects, mean plasma adrenaline level was higher in group A (0.23 +/- 0.09 nmol/L) than in group B (0.15 +/- 0.12) but the difference was not statistically significant. Blood group A individuals responded to a stressful situation with higher levels of cortisol, and possibly of adrenaline. These observations tend to support findings of previous studies demonstrating a high risk of diseases related to stress (coronary heart diseases and gastrointestinal diseases), in men with A blood group.

Chlordiazepoxide, go-nogo successive discrimination and brain biogenic amines in cats.

Chlordiazepoxide (CDP; 0.4 mg/kg/day, per os) was administered to cats during either the acquisition (CDP 21-22 days) of a go-nogo successive discrimination task (SD) or the performance (CDP 10 days) of the previously learned SD task. Endogenous levels of serotonin, 5-hydroxyindoleacetic acid, noradrenaline and dopamine were assayed in 12 brain areas, in trained as well as in untrained cats. This study has shown that (1) CDP strongly impaired the acquisition but not performance of the SD task, revealing a dissociation of the effects of CDP on these two stages of training; (2) the CDP administration, as well as the SD training, produced regional changes in brain levels of biogenic amines, suggesting the involvement of particular monoaminergic neurons in the behavioral effects of CDP and in operant behavior; and (3) in particular brain areas, interactions were observed between the effects of the SD training and those of the CDP administration on monoamines, indicating that the behavioral state may interfere with the neurochemical effects of CDP.

Biochemical properties and kinetic parameters of monoamine oxydase in human seminal plasma.

The present study demonstrates that monoamine oxydase (MAO) is present in the human seminal plasma and that its activity was higher in infertile than in fertile men. The Km and Vmax of the enzyme were, respectively, 3.0 X 10(-3) M and 625 eta mol of deaminated 5-HT mg of prot-1 min-1. The enzyme was activated by pyridoxal-5' phosphate but inhibited by usual MAO inhibitors such as pargyline-HCl, iproniazid-PO4 and clorgyline. These findings suggest a relationship between the synthesis and degradation of biogenic amines and testicular function.

Dietary intake data: usefulness and limitations.

In past decades, the main objectives of nutrition surveys were to define nutritional status at the time of the study, to cross-sectionally describe dietary patterns of consumption and food preparation practices, and to identify areas for improvement. Nowadays, the need for ongoing evaluation of nutritional status of individuals and of population is gaining recognition. The present paper, in discussing usefulness and limitations of dietary intake data, will focus on the importance of considering nutrition as a component of overall quality of life as well as a discipline that gains to be integrated to other disciplinary fields such as medicine, biochemistry, immunology, anthropometry and agribusiness. In this respect, the present paper will discuss the methodology of dietary data collection, the various uses of dietary data collected at the individual, community and general population levels and finally will try to define new vistas in regard to emphasis and tools for future dietary intake studies.

Determination of mono-, di- and polyamines in foods using a single-column amino acid auto-analyzer.

A fully automated, rapid and sensitive method was developed to analyze fourteen different biogenic amines in food. Using a Technicon C4 ion-exchange resin column (20 m X 0.5 cm), adapted to an automatic Technicon TSM amino acid analyzer, the following amines were separated and quantified: adrenaline, noradrenaline, 1,3-diaminopropane, putrescine, cadaverine, histamine, spermidine, dopamine, spermine, agmatine, tyramine, serotonin, phenethylamine and tryptamine. Five buffers were required to elute the amines using a gradient of pH from 5.6 to 12.7; the column temperature was maintained at 65 degrees C. The method was also assayed on ground beef, cheese and wine samples. Amines from cheese and ground beef samples were extracted with 0.6 M perchloric acid. No extraction of wine samples was necessary.

Effects of chlordiazepoxide administration on biogenic amines in cat brain.

Cats underwent treatment with chlordiazepoxide hydrochloride (0.4, 10.0, and 20.0 mg/kg per os), for 7 consecutive days, and were killed 18 h after the last administration. The endogenous levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), noradrenaline (NA), and dopamine (DA) were assayed in 12 brain areas. Few effects on 5-HT, 5-HIAA, and NA content and on the 5-HT:5-HIAA ratio were observed with a 0.4 mg/kg treatment. These changes were localized in the piriform lobe (amygdala), hippocampus, mesencephalon, and mesencephalon raphe nuclei. Moreover, the DA concentration was not affected. The changes produced by 10.0 and 20.0 mg/kg chlordiazepoxide treatments were extended to many more structures, including the limbic system, brainstem, diencephalon, and neostriatum with respect to 5-HT, 5-HIAA, and NA content and also to DA levels. The changes observed after the three doses generally included an increased 5-HT content, a decreased 5-HIAA level, a high 5-HT:5-HIAA ratio, and increased NA and DA concentrations. However, in some structures, a decreased NA content and an increased 5-HIAA level were found. The present results suggest that administration of chlordiazepoxide for 7 consecutive days in cats produces regional changes in the content of endogenous biogenic amines in the central nervous system (CNS) at low doses; much more extended effects are produced at high doses. These findings are in agreement with a reducing effect of benzodiazepines on the turnover and release of biogenic amines in the CNS, but also suggest that certain discrete areas are more involved in these changes, thus dissociating them from the rest of the brain.

Effects of chlordiazepoxide on acquisition and performance of a go-no go successive discrimination task, and on brain biogenic amines in cats.

1. Normal and chlordiazepoxide (0.4 mg/kg/day, per os) treated cats were trained on a symmetric go-no go successive discrimination task with positive reinforcement. 2. The treatment impaired the acquisition, but not the performance once the task was learned. 3. Serotonin, 5-hydroxyindoleacetic acid and noradrenaline were assayed in 12 brain areas in trained cats, and in normal and treated cats which did not undergo the training. 4. The treatment produced localized changes in the serotonin: 5-hydroxyindoleacetic acid ratio and the noradrenaline level; these effects were modified by the training. 5. The drug induced an acquisition deficit rather than a general behavioral disinhibition, and produced neurochemical effects which were dependent upon the brain area, the treatment duration and the behavioral state.