Purification and characterization of {delta}-aminolevulinic acid dehydratase from Chlorella regularis

-Aminolevulinic acid dehydratase (-aminolevulinic acid hydrolyaseEC 4.2.1.24 [EC] ) which catalyzes the formation ofporphobilinogenfrom two molecules of -aminolevulinic acid (ALA) was purifiedfrom Chlorella regularis 737-fold by acetone and ammonium sulfatefractionations, DEAE-cellulose column chromatography, and SephadexG-200 gel filtration. The enzyme had an optimum pH of 8.5 inTris-HCl buffer and required either Mg²⁺ or Mn²⁺ for its maximumactivity. The Km values for Mg²⁺, Mn²⁺ and ALA were 15 µM,10µM, and 0.5 mM, respectively. The enzyme was not activatedby thiol compounds, but was inhibited by p-chloromercuribenzoate.The molecular weight estimated by gel filtration was 316,000and the isoelectric point was 5.25. (Received October 18, 1978; )     

LEVELS OF NOREPINEPHRINE AND DOPAMINE IN MOUSE BRAIN REGIONS FOLLOWING MICROWAVE INACTIVATION-RAPID POST-MORTEM DEGRADATION OF STRIATAL DOPAMINE IN DECAPITATED ANIMALS

—The effects of 2 methods of killing on norepinephrine and dopamine in mouse brain regions were examined. One method utilized decapitation, while the other method utilized heating with microwave irradiation concentrated on the head. The norepinephrine and dopamine contents of the cerebellum, medulla-pons, midbrain, diencephalon, hippocampus, corpus striatum, and cerebral cortex were determined by methods using liquid chromatography with electrochemical detection. Dopamine content in striatum was also quantitated by the method of gas chromatography with mass fragmentography. A significantly lower value for decapitated animals, as compared to the microwave heated group, was found only for dopamine exclusively in the striatum. Activities of the enzymes tyrosine hydroxylase, DO PA decarboxylase, monoamine oxidase, and catechol-o-methyltransferase in the striatum were also examined. These enzymes were totally inactivated by the microwave heating, except catechol-o-methyltransferase which was decreased approx 80%. These results support either (1) the existence of a substantial pool of dopamine in the striatum with a very rapid turnover rate or (2) a decapitation-related release and destruction of striatal dopamine. Measurements of 3-methoxytyramine in the striatum exhibit post-mortem increases corresponding to the decreases of dopamine. Use of the rapid tissue enzyme inactivation technique suggests that in vivo levels of this O-methylated dopamine metabolite are an order of magnitude lower than the results normally obtained after killing by decapitation.     

Oxantel is an N-type (methyridine and nicotine) agonist not an L-type (levamisole and pyrantel) agonist: classification of cholinergic anthelmintics in Ascaris

Three pharmacological subtypes of cholinergic receptors have been distinguished in Ascaris suum using a muscle contraction assay and classical pharmacological techniques. The receptor subtypes are: a B-subtype (sensitive to bephenium); an L-subtype (sensitive to levamisole and pyrantel); and an N-subtype (sensitive to nicotine and methyridine). Oxantel is a cholinergic anthelmintic that was first introduced for the treatment of whipworm, Trichuris, infections in children. Here, we compare the subtype selectivity of oxantel with thenium and other cholinergic anthelmintics. We used the A. suum assay to derive pA(2) values for the agonists: oxantel, thenium, bephenium, levamisole, pyrantel, nicotine and methyridine with the antagonists: paraherquamide, 2-desoxyparaherquamide and methyllycaconitine. pA(2) values, rather than pK(B) values, were determined for all agonists when it was found that Schild slopes for some agonists were significantly less than 1.0. The pA(2) of oxantel was 6.58+/-0.25 for paraherquamide; 5.39+/-0.28 for 2-desoxyparaherquamide; 7.01+/-0.19 for methyllycaconitine. Comparison of pA(2) values using cluster analysis showed that oxantel was grouped with nicotine and methyridine, the N-subtype agonists. Thenium had pA(2)s of 7.84+/-0.41 for paraherquamide; 5.52+/-0.50 for 2-desoxyparaherquamide; 6.33+/-0.19 for methyllycaconitine. Cluster analysis placed thenium between the L-subtype agonists and the B-subtype agonist. The therapeutic significance of classification of cholinergic anthelmintics is discussed. Combination of oxantel and pyrantel would have therapeutic advantages, covering N- and L-subtypes, and so increasing spectrum of action and reducing the potential for development of resistance. Our results predict that oxantel may remain effective in some nematode isolates that have become levamisole- and pyrantel-resistant.     

Secular change in body height and cephalic index of Croatian medical students (University of Rijeka)

An investigation of body height and cephalic measurements was performed among five groups of first-year medical students of the University of Rijeka School of Medicine (Rijeka, Croatia). Body height and different cephalic measurements showed normal distribution, both in male and female students. Differences between measured variables were statistically analyzed by ANOVA. No significant difference with regard to year of birth was found in either males or females. The cephalic index showed no statistically significant difference between sexes or with regard to body height, while head breadth and length correlated significantly with birth year and body height, both in males and females. Head breadth decreased within the study period, while head length increased. Results were compared with those of similar studies from the mid-20th century. Student's t-test showed a significant change of cephalic indices and other head measurements, but not of body height, in males. The frequency difference between various head shapes was tested using the chi-square test. A significant increase of dolichocephalic and mesocephalic and a significant decrease of brachycephalic head shape were found in both sexes. These results suggest a continuity of the debrachycephalization process observed in our population at the past midcentury.     

Anthropological determinants of success in young judoists

There is an evident lack of experimental studies dealing with the anthropological determinants of success in judo, especially concerning young athletes. In this paper we have attempted to establish some of the anthropological factors potentially related to judo competition achievement in 15-16 year old athletes. A set of 14 anthropometric and 12 motor-endurance variables was tested on the best young judoists in Croatia (all males, N = 34). All the subjects had competed on the National Championship (NC), and the criterion was defined according to their final NC achievement. Factor analysis and discriminant canonical analysis (DISCRA) were calculated separately on the motor-endurance and anthropometric status variables. DISCRA showed the successful judoists as dominant in strength and endurance status. In contrast, there were no significant differences in the anthropometric dimensions regarding any differentiation between the more and less successful athletes. Possible explanations are discussed.     

Morphological structures of elite Karateka and their impact on technical and fighting efficiency

The aim of the study was to identify the morphological structures that determine achievement of top results in karate. The study included a sample of 85 karateka competing as senior category athletes within the Croatian Karate Society, aged 18-29 years. Sixteen morphological parameters were used; technical efficiency was assessed using 8 variables, i.e. evaluation of particular karate techniques, whereas fighting efficiency was estimated on the basis of results achieved at a number of contests. Factorial analysis of morphological space revealed the presence of four major factors: factor of muscle mass, followed by skeleton transverse dimensionality, factor of skeleton longitudinal dimensionality, factor of subcutaneous adipose tissue, and factor of shoulder width determining optimal trunk proportions (athlete type) in karateka. Regression analysis showed the isolated group of morphological factors to significantly determine both technical and fighting efficiency of karate athletes. Generally, skeleton longitudinality and muscle mass, followed by skeleton transverse dimensionality were found to exert favorable effects, and adipose tissue unfavorable effects according to both criteria. Also, each individual morphological factor influenced the performance of every karate technique applied. Adipose tissue had greatest unfavorable impact, whereas shoulder width and muscle mass had favorable impact on the performance of arm techniques (kicking). Considering performance of leg techniques, skeleton longitudinality had highest favorable impact, and adipose tissue greatest unfavorable impact. Of the techniques applied, combined karate kicks, i.e. jaku zuki-mawashi geri and kizame zuki-jaku zuki were found to be the best predictors of fighting efficiency.     

Purification and Characterization of 4,5-Dioxovalerate Reductase (NADPH) from Chlorella regularis

Two types of 4,5-dioxovalerate reductases (NADPH) were partiallypurified and characterized from green alga, Chlorella regularis.The enzyme was separated by DEAE-Sephacel chromatography intotwo peaks: type I (first peak) and type II (second peak). Theactivity ratio of the type II to type I enzyme varied between5 to 7 with a starting cell material. Both enzymes had the samepH optimum at 6.0 and pI value of 4.9. The molecular weightestimated by gel filtration was 33,000 for type I and 99,000for type II enzyme. Both enzymes used only NADPH, but were notspecific for 4,5-dioxovaleric acid (DOVA). Type I enzyme reducedglyoxylate 68-fold faster than DOVA, whereas type II enzymeacted more specifically on a variety of aldehydes than DOVA.It is suggested that these enzymes may not function primarilyas NADPH-DOVA reductases in the metabolic pathway of DOVA. (Received June 15, 1985; Accepted October 14, 1985)     

Ethanol potentiates the effect of gamma-aminobutyric acid on medial vestibular nucleus neurons responding to horizontal rotation

Electrophysiological studies were performed to determine whether or not ethanol potentiates the inhibitory effects of gamma-aminobutyric acid (GABA) on medial vestibular nucleus (MVN) neurons responding to horizontal sinusoidal rotation using alpha-chloralose anesthetized cats. The MVN neurons were classified into types I, II, III and IV neurons according to the responses to the horizontal rotation of the animal placed on the turntable in directions ipsilateral and contralateral to the recording site. In addition, the effects of ethanol and GABA on type I neurons were also examined. Micro-osmotic application of ethanol up to 100 nA did not affect the spontaneous firing or the rotation-induced increase in firing of type I neurons. However, the inhibitory effects of GABA up to 50 nA on the rotation-induced increase in firing were potentiated during simultaneous application of ethanol up to 100 nA. This potentiated inhibition was blocked by iontophoretic application of bicuculline (25-150 nA) and picrotoxin (45-150 nA). These results suggest that ethanol potentiates the inhibitory effects of GABA on MVN type I neurons by acting on the GABA receptor and/or receptor-coupled chloride ion channel.     

Dopamine D-2 receptor-mediated excitation of caudate nucleus neurons from the substantia nigra

Microiontophoretic studies using cats anesthetized with alpha-chloralose were performed to elucidate whether the excitatory response of caudate nucleus (CN) neurons upon stimulation of the pars compacta of the substantia nigra (SN) is mediated by the dopamine D-1 or D-2 receptor. There were rare convergent inputs from the SN and motor cortex (MC) in the CN neurons. Iontophoretic application of haloperidol and domperidone (dopamine D-2 receptor antagonist) produced dose-dependent inhibition of spikes elicited by SN stimulation in 25 of 42 and 50 of 82 CN neurons, respectively, however, no alterations of spikes elicited by MC stimulation occurred in any 11 neurons tested. Iontophoretically applied SCH 23390 (D-1 antagonist) did not inhibit the SN-induced spikes in any CN neurons, of which spikes were inhibited by domperidone. These results suggest that the SN-induced spikes are mediated by dopamine, which acts on postsynaptic D-2 receptors.     

Nonequilibrium Dissipation-free Transport in F1-ATPase and the Thermodynamic Role of Asymmetric Allosterism

F₁-ATPase (or F₁), the highly efficient and reversible biochemical engine, has motivated physicists as well as biologists to imagine the design principles governing machines in the fluctuating world. Recent experiments have clarified yet another interesting property of F₁; the dissipative heat inside the motor is very small, irrespective of the velocity of rotation and energy transport. Conceptual interest is devoted to the fact that the amount of internal dissipation is not simply determined by the sequence of equilibrium pictures, but also relies on the rotational-angular dependence of nucleotide affinity, which is a truly nonequilibrium aspect. We propose that the totally asymmetric allosteric model (TASAM), where adenosine triphosphate (ATP) binding to F₁ is assumed to have low dependence on the angle of the rotating shaft, produces results that are most consistent with the experiments. Theoretical analysis proves the crucial role of two time scales in the model, which explains the universal mechanism to produce the internal dissipation-free feature. The model reproduces the characteristic torque dependence of the rotational velocity of F₁ and predicts that the internal dissipation upon the ATP synthesis direction rotation becomes large at the low nucleotide condition.