Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats

Summary. We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n=7), thyroidectomized rats (n=6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n=7), thyroidectomized rats with deficient diet (n=9). Homocysteine was decreased in operated rats (2.6±1.01 vs. 4.05±1.0mol/L, P=0.02) and increased in deficient diet rats (29.56±4.52 vs. 4.05±1.0mol/L, P=0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P=0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine--synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine--synthase activity.     

Metabolic parameters of epilepsy: adjuncts to established antiepileptic drug therapy

Hughlings Jackson at the turn of the century defined epilepsy as a disorder originating in a morbid nutrition of the neuron. With the advances in modern neurochemistry, it is becoming increasingly clear that a chronic seizure predisposition or a lowering of the brain's discharge threshold can be demarcated by a number of biochemical markers. They include a tendency for an increased release of glutamate with or without GABAergic impairment, (intra)neural tissue alterations in water redistribution/osmolarity or other distortions of the cytoarchitecture, and an elevation of ionic calcium inside the cell. These changes are dominantly shared parameters of the seizure prone brain. Magnetic resonance spectroscopy (MRS) shows that cerebral levels of glutamate + glutamine (Glx) are increased interictally in epileptogenic regions in human partial epilepsy; other findings using this technique suggest damage to (cellular/mitochondrial) membranes, denoted by N-acetyl-aspartic acid (NAA) changes and a decreased energy capability (1). The merging of previous in vitro and ex vivo findings in neurophysiology and neurochemistry with magnetic resonance spectroscopy technology provides a powerful new methodology to interpret and to obtain clinical insight into the metabolic alterations that underlie an epileptogenic process. In this review some of these basic neurochemical and electrophysiological mechanisms are discussed. In addition, certain adjuncts to established antiepileptic drug therapy are suggested in the hope that over the long term they may help in correcting the primary metabolic deficits.     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.     

The filtration apparatus of Cladocera: Filter mesh-sizes and their implications on food selectivity

Summary The filtering apparatus of eleven Cladoceran species was studied. The distances between the setulae, which act as filters, were measured. Among adult individuals, they vary from 0.2 m in Diaphanosoma brachyurum to 4.7 m in Sida crystallina. Species can be grouped according to the mesh-sizes, as fine mesh filter-feeders: Diaphanosoma brachyurum, Ceriodaphnia quadrangula, Chydorus sphaericus, Daphnia cucullata and Daphnia magna; medium mesh filter-feeders: Daphnia galeata, D. hyalina. D. pulicaria, Bosmina coregoni, and coarse mesh filter-feeders: Holopedium gibberum and Sida crystallina. In Daphnia hyalina, the distances between setulae increase from 0.3–0.4 m in small juveniles, to 0.8–2.0 m in adults. In Daphnia magna, the mesh-size of the filter does not increase significantly with growth. There is good evidence that the relative abundance of the filter-feeding types varies with the trophic state of the lake. In oligotrophic lakes the coarse mesh filter-feeders usually dominate throughout the year. The seasonal succession of zooplankton species in eutrophic lakes can be interpreted as a succession of feeding types; during winter coarse mesh filter-feeders dominate, while fine mesh filter-feeders are most abundant during summer phytoplankton blooms. Our results support the hypothesis that the species composition of filter-feeding zooplankton is strongly influenced by the amount of suspended bacteria which are available as food only for filter-feeding species with fine meshes.     

UDP-galactose:lactosylceramide α-galactosyltransferase activity in human placenta

The activity of UDP-Gal: LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn²⁺ at pH 6.3, and the reaction product was susceptible to -galactosidase.Abbreviations LacCer lactosylceramide (Gal1-4Glc1-1Cer) - Gb₃ globotriaosylceramide (Gal1-4Gal1-4Glc1-1Cer) - Gb₄ globoside (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer) - TLC thin-layer chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance - EDTA ethylenediamine tetraacetic acid - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate     

Acute effects of sodium valproate and γ-vinyl gaba on regional amino acid metabolism in the rat brain: Incorporation of 2-[14C]glucose into amino acids

Amino acid concentrations have been determined in rat brain regions (cortex, striatum, cerebellum, and hippocampus) by HPLC after administration of acute anticonvulsant doses of sodium valproate (400 mg/kg, i.p.) and -vinyl-GABA (1g/kg, i.p.). After valproate administration the GABA level increases only in the cortex; aspartic acid concentration decreases in the cortex and hippocampus, and glutamic acid decreases in the hippocampus and striatum and increases in the cortex and cerebellum. There are no changes in the concentrations of glutamine, taurine, glycine, serine, and alanine following valproate administration. Only the GABA level increases in all the regions after -vinyl-GABA administration. Cortical analyses 2, 4 and 10 minutes after pulse labeling with 2-[¹⁴C]glucose, i.v., shown no change in the rate of cortical glucose utilization in the valproate treated group. The rate of labeling of glutamic acid is also unchanged, but the rate of labeling of GABA is reduced following valproate administration. After -vinyl-GABA administration there is no change in the rate of labeling of GABA. These biochemical findings can be interpreted in terms of a primary anticonvulsant action of valproate on membrane receptors with secondary effects on the metabolism of amino acid neurotransmitters. This contrasts with the primary action of -vinyl-GABA on GABA-transaminase activity.This paper is dedicated to Dr. Derek Richter on his sevety-fifth birthday     

Body temperature responses of Savanna Brown goat to the harmattan and hot-dry season

Rectal and vaginal temperature responses of the Savanna Brown goat indigenous to the Nigerian guinea savanna were determined during the harmattan and the hot-dry season. Measurements were made at 06:00h and at 14:00h after 8h exposure to field conditions. At the 06:00h measurements during the harmattan, all animals were observed to shiver. A significant (P<0.01) positive correlation was found between rectal (T_re) and vaginal temperatures. During the harmattan, mean T_re was 38.2C at 06:00h and 39.7C at 14:00h; the mean difference, T_re was 1.5C. During the hot-dry season, T_re at 06:00h was 38.1C, and at 14:00h, 38.7; T_re was 0.6C. It is concluded that the harmattan is thermally more stressful than the hot-dry season and that passive thermolability may not be an important mechanism in the Savanna Brown goat in adaptation to thermal stress.     

A role for guanidino compounds in the brain

Guanidino compounds of guanidinoethanesulfonic acid, guanidinoacetic acid, guanidinosuccinic acid, N-acetylarginine, -guanidinopropionic acid, creatinine, -guanidinobutyric acid, arginine, guanidine, methylguanidine, homoarginine and -guanidinoglutaric acid are present in the mammalian brain. These guanidino compounds except for arginine and guanidine induce seizures and convulsions in rat, rabbit and cat by intracisternal injection.Hirudonine, audonine, -keto--guanidinovaleric acid, N,N-dibenzoylguanidine and phenylethylguanidine are also convulsants. Levels of creatinine, guanidinoethanesulfonic acid, creatinine, guanidinoacetic acid and methylguanidine in animal brain were changed at pre- and during convulsions induced by pentylentetrazol, amygdala kindling, iron-induced epileptogenesis and so on. These convulsions are thought to be due to depressed functions of serotonergic neurons and accumulated free radicals.Arginine is a substrate of nitric oxide production by nitric oxide synthase. -Guanidinoglutaric acid is a generator of superoxide, hydroxyl radicals and nitric oxide, and induced C6 glial cell death. On the other hand, aminoguanidine is a free radical scavenger. Energy formation by creatine metabolism may inhibit apoptosis induced by pathogenesis. Free radical generation/reaction and energy generation by guanidino compounds must be important key role in the brain.     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

Metabolic manipulation of neural tissue to counter the hypersynchronous excitation of migraine and epilepsy

Very prominent in the large biochemical data bank on epilepsy, is the almost universal finding that a familial or environmental predisposition towards epilepsy, as well as the earliest signs preceding other forms of hypersynchronous excitation, coincide with an altered glutamate metabolism. Hence, it has become increasingly apparent that glutamate occupies a central position in the development of epilepsy or in the onset of a migraine incident. The importance of glutamate is explained by a variety of functions in the CNS: as a dominant constituent of many proteins, by its intermediary role in linking energy metabolism to that of many other amino acids, and as the virtually exclusive precursor of GABA. Moreover, glutamate serves as the primary substrate in ammonia detoxification and the product, glutamine, actively participates in CSF water homeostasis. Finally, by its direct electrophysiological and metabolic actions on neurons and glia, via at least four distinct types of receptor proteins, glutamate is implicated in a number of critical mechanisms of information. These include neuronal excitatory modulation, intracellular Ca²⁺ redistribution, and key metabolic (phosphorylation) mechanisms. The phenomena, when exaggerated due to excessive extracellular glutamate levels, may cause pathological effects such as hypersynchrony-epilepsy, Spreading Depression-migraine, high internal Ca²⁺-damage, impaired phosphorylation/dephosphorylation-necrosis, among others. Not surprising therefore that severe epilepsy may eventually cause CNS cytoarchitectual and metabolic damage, or conversely, that neural tissue trauma not infrequently gives rise to epilepsy many years later. Both conditions are associated with a persistent, excessive leakage or release of glutamate into the extracellular milieu. An electrophysiological and neurochemical commonality between migraine and epilepsy has also been noted. A specific nutritional supplement is proposed, consisting of a combination of taurine (100 mg),l-leucine (75 mg), (acetyl-)l-carnitine (25 mg), and zinc gluconate (10 mg) which is aimed at, principally, normalizing internal Ca²⁺ ionization, and removal of extracellular glutamate by glial amidation to glutamine.Special issue dedicated to Dr. Claude Baxter.     

Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry

Summary The cyclization of a linear dynorphin A (Dyn A) analogue to give the lactam derivative cyclo[d-Asp², Dap⁵]Dyn A(1–13)NH₂ (where Dap=,-diaminopropionic acid) was studied to evaluate the usefulness of different coupling reagents for side chain to side chain lactam formation. This cyclization proved to be difficult and yielded substantial byproducts that varied depending upon the activating reagent used. On-line HPLC-ion spray mass spectrometry was more practical and useful than conventional HPLC alone for characterizing the products of these cyclization reactions. Peptide byproducts could be identified from the series of multiply charged ions observed, even when some of these peptides eluted from the HPLC with similar retention times. In addition to the desired cyclic peptide, the peptide byproducts observed following the cyclization using BOP (benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate) were the linear peptide, the cyclic dimeric peptide and the linear peptide resulting from aspartimide rearrangement. The peptide byproducts obtained following cyclization using HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HAPyU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate) were predominantly linear tetramethylguanidinium (Tmg) and dipyrrolidinylguanidinium (Dpg) derivatives resulting from alkylation of the side chain of Dap by HATU and HAPyU, respectively; in addition to monomeric guanidinium derivatives, dimeric and aspartimide-containing peptides were also produced. Peptide sequencing by ion spray tandem mass spectrometry was performed to confirm the structure of both pure peptides and peptide byproducts in the crude samples. A unique fragmentation for the ,-bond of the Dap side chain was demonstrated and could be used to identify linear peptide byproducts. The distinctive fragment ions from this cleavage were also observed for the peptides containing the Tmg and Dpg functionalities on the Dap side chain.     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

The two β-oxidation sites in pea cotyledons

Two sites for -oxidation of fatty acids in pea (Pisum sativum L.) cotyledons exist. One site is the microbody, the other the mitochondrion. Mitochondrial -oxidation of fatty acids is carnitine-dependent. The fatty acid permeates the membrane as palmitoylcarnitine which is formed from cytosolic-side palmitoyl-CoA by a carnitine palmitoyltransferase located on the exterior face of the inner mitochondrial membrane as a peripheral protein. A single-gated pore integral membrane translocator is proposed to exchange the palmitoylcarnitine for carnitine or acetylcarnitine across the membrane. An internal (matrix side) carnitine palmitoyltransferase then reforms palmitoyl-CoA which enters -oxidation and subsequently the tricarboxylic-acid cycle.     

Comparison of Fusarium acuminatum and Fusarium culmorum isolates by means of tandem-crossed immunoelectrophoresis

F. acuminatum and F. culmorum strains were compared by means of tandemcrossed immunoelectrophoresis in order to estimate the possiblities of serological classification in Fusarium sections Gibbosum and Discolor. On the basis of qualitative similarity the two species could be distinguished well. By the use of anti-F. acuminatum serum a similarity of S_SM=0.52 was found between F. acuminatum and F. culmorum, but the S_SM coefficient reached a value of 0.67 when the anti-F. culmorum serum was tested. This asymmetric nature of the qualitative similarity is discussed. In the majority of cases the quantitative differences of the common antigens did not allow differentiation between the species.     

Amino acid sequence of the alpha- and beta-globin chains of the Hiroo sea snake (Laticauda laticaudata)

We determined the hemoglobin complete amino acid sequences of the Hiroo sea snake (Laticaudia laticuada) from the intact globin chain, enzymatically digested fragments, and chemical cleavage fragments to analyze molecular evolution for classification of the sea snake. The Hiroo sea snake has two hemoglobin components, Hb-I and Hb-II, which contain different - and -chains, respectively. This is the first report of the complete primary structure of a snake hemoglobin. The sequences were compared with those of other reptilian hemoglobins. Amino acid replacements at positions critical for structure and physiological role of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors at -globins seemed to be fullfilled.     

Biological and physico-chemical properties of an atypical mutant of tobacco mosaic virus

Summary A mutant of tobacco mosaic virus (TMV) was isolated from a population of TMV-RNA molecules inactivated to 0.1% level of survival by treatment with nitrous acid This mutant is poorly transported in Samsun tobacco and induces chlorotic spots, oak-leaf patterns, ringspots and necrosis. Rate zonal sucrose density-gradient and equilibrium sedimentations in sucrose and CsCl indicate that the purified mutant preparations contain a heterogenous population of particles including some free coat protein. These vary in length, but approximately 5–10% are infectious rods of standard (15×300 nm) dimensions. Purified mutant RNA contain an overabundance of fragmented and a few infectious molecules. Compared to the parent strain, the mutant capsid protein contains two additional residues of arginine and one of glycine, but lacks one residue each of glutamic acid (or glutamine), serine and threonine. These observations and other data based on temperature sensitivity, exposure to urea and serology indicate that the mutant particles are inherently unstable and break during the purification procedure.     

The demonstration of phospholipids in certain cells in neurosecretory nuclei in the rat with Baker's acid haematein after fixation in glutaraldehyde-formol-calcium

Summary With Baker's acid haematein test certain ganglion cells in the brain, their processes and, at some sites, glial cells around blood vessels stain dark blue. This article describes a study of the Baker-positive cells which occur in and around the neurosecretory nuclei. By substituting formol-calcium fixation with glutaraldehyde-formol-calcium fixation shrinkage in brain tissue is completely avoided. If such fixation is used the argument that positive staining of ganglion cells with Baker's method only indicates that these are shrunken neurons can no longer be maintained. A comparative histological study, especially of Baker's technique and controlled chromation (Elftman) showed that the Baker-positive cells contain a phospholipid, probably bound to a protein, as a labile compound, which is easily lost. We found that to immobilize and localize this labile compound in the ganglion cells the technique of fixation and the pH during chromation (which should be around 3.8) are of fundamental importance. Only under these conditions is the complex sufficiently immobilized to allow of its demonstration with acid haematein. These requirements are now completely met if Baker's acid haematein technique is used. The article stresses that only prefixed and chromated frozen sections can be used for this method, thus avoiding shrinkage and non-specific staining of proteins. The modified Baker method as used by us gives constant and reproducible staining and is described in this article. The functional significance of the Baker-positive reaction in some ganglion cells in the n. s. nuclei or glial cells around blood vessels is not dealt with in this article.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Intranuclear inclusions in pericytes of the hypothalamus of the rat

Summary This paper deals with the ultrastructure of two types of intranuclear inclusions, nuclear bodies and membranous lamellar bodies, present in hypothalamic pericytes of intact adult rats. The nuclear bodies exhibited simple and granular forms, whereas the membranous lamellar bodies were entirely made up of myelin-like membrane whorls.The occurrence of these bodies in nuclei of pericytes has never been previously reported. The origin and functional meaning of such structures is discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.