Molecular Characterization of Aldolase from Heterodera glycines and Globodera rostochiensis.

Fructose-bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in glycolysis. We have characterized full-length coding sequences for aldolase genes from the cyst nematodes Heterodera glycines and Globodera rostochiensis, the first for any plant-parasitic nematode. Nucleotide homology is high (83% identity), and the respective sequences encode 40 kDa proteins with 89% amino acid identity. Genomic sequences contain six introns located at identical positions in both genes. Intron 4 in the H. glycines gene is >500 bp. Partial genomic sequences determined for seven other cyst nematode species reveal that the large fourth intron is characteristic of Heterodera but not Globodera aldolase genes. Total aldolase-like specific activity in homogenates from H. glycines was 2-fold lower than in either Caenorhabditis elegans or Panagrellus redivivus (P = 0.001). Activity in H. glycines samples was higher in juvenile stages than in adults (P = 0.003). Heterodera glycines aldolase has Km = 41 microM and is inhibited by treatment with carboxypeptidase A or sodium borohydride.

Comparison of FaRP immunoreactivity in free-living nematodes and in the plant-parasitic nematode Heterodera glycines.

The family of FMRFamide-related peptides (FaRPs) is widely distributed among invertebrates, where the peptides serve as neuromodulators. Published reports indicate that numerous FaRP sequences exist in free-living and animal parasitic nematodes. Using a FMRFamide ELISA, FaRP immunoreactivity was detected in extracts of the soybean cyst nematode, Heterodera glycines, in both sexes and at all developmental stages. HPLC-ELISA results revealed a number of immunoreactive components in H. glycines preparations, and a comparison with extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus showed significant qualitative differences in FaRP immunoreactivity between the plant parasite and the two free-living nematodes. Total and specific immunoreactivities varied during H. glycines development, with the highest specific activity in juveniles and males, and the highest total activity in mature females. Total female immunoreactivity was located primarily within the mature eggs. A significant portion, however, was associated with the female body, perhaps with egg laying.

In vitro metabolism of an insect neuropeptide by neural membrane preparations from Lymantria dispar.

Neural membrane fractions, prepared from brain-subesophageal ganglion complexes of the adult lepidopteran Lymantria dispar, contain at least two peptidases capable of metabolizing locust adipokinetic hormone-I in vitro. The initial fragments, pGlu1-Leu2-Asn3 and Phe4-Thr5-Pro6-Asn7-Trp8-Gly9-Thr10, result from the action of an endopeptidase with properties similar to those reported for neutral metalloendopeptidase in Schistocerca gregaria and mammalian endopeptidase 24.11. The heptapeptide is further degraded by an aminopeptidase that exhibits kinetic properties similar to those described for aminopeptidase 3.4.11.2. These enzymes appear to be responsible for the first two steps in AKH catabolism in L. dispar.

FMRFamide-like Immunoactivity in Heterodera glycines (Nemata: Tylenchida).

Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes.

[Analysis of subpopulations of peripheral blood T-lymphocytes of schizophrenic patients]. / Analiz subpopuliatsii T-limfotsitov perifericheskoi krovi bol'nykh shizofreniei.

The reaction of lymphocytes to Con A and PHA and combined action of these 2 T-mitogens in the peripheral blood was studied in schizophrenic patients (25 cases) and normal donors (21 cases). It was demonstrated that study of lymphocyte function in the peripheral blood with the aid of the 2 mitogens makes it possible to differentiate more distinctly the lymphocytes of schizophrenic patients and normals. It was established that the lymphocyte subpopulation capable of responding to both mitogens used is 4 times less in schizophrenics than in normal donors. The proportion of cells reacting only to one of these mitogens is significantly higher than in normals. The total content of mitogen-sensitive T-lymphocytes in schizophrenic patients and normal donors is approximately the same and equals about 40% of the whole lymphocyte population in the peripheral blood.

[Role of schizophrenic patients' serum in the altered osmotic resistance of their lymphocytes]. / Rol' syvorotki krovi bol'nykh shizofreniei v izmen ennoí osmoticheskoi rezistentnosti limfotsitov étikh bol'nykh.

The resistance of peripheral blood lymphocytes was estimated in normal subjects and schizophrenic patients with reference to hypoosmotic effects. It was shown that in normal subjects about 20% of the lymphocytes were slightly resistant. In 60% of the schizophrenic patients the fraction of the slightly resistant lymphocytes was found to be sharply lowered. The content of those cells in the blood of patients with different forms of the disease course was different: patients with the periodic form showed the minimal, and patients with continuously progressing form the maximal deviation from normal. Patients with the schubweise form occupied an intermediate place. Incubation of the lymphocytes of the healthy donors in the serum of schizophrenic patients led to selective destruction of the donor' slightly resistant cells. A statistically significant correlation between the destructive capacity of the patients' serum and the lowering of the content of the slightly resistant lymphocytes in those patients was observed.

[Dependence of the peripheral blood lymphocyte reaction of schizophrenia patients to concanavalin A stimulation on the clinical parameters of the pathological process]. / Zavisimost' reaktsii limfotsitov perifericheskoi krovi bol'nykh shizofreniei na stimuliatsiiu konkanavalinom A ot nekotorykh klinicheskikh parametrov patologicheskogo protsessa.

Studies were performed on an equivalent age group of schizophrenic patients and normal donors to detect the lymphocyte reaction of the peripheral blood to concanavalin A stimulation. It was demonstrated, that in the blood of schizophrenic patients, the part of lymphocytes capable of reacting to stimulation, is 2,3 times less than in the group of normals. The studies showed that the capability of cells to react to stimulation does not depend upon the duration of the disease, the duration of the postmanifest or initial periods, but differs in patients with different forms of schizophrenia. A significant negative correlation was demonstrated between the severity of positive disorders and the lymphocyte response to stimulation.

[Dynamics of the lipid peroxidation process in the body of patients with manic-depressive psychosis]. / Dinamika protsessov perekisnogo okisleniia lipidov v organizme bol'nykh maniakal'no-depressivnym psikhozom.

Using gas chromatography the authors have studied the time course of lipid peroxidation (LPO) in patients with manic depressive psychosis (a depressive phase) during a course of antidepressive therapy. The results indicate that the levels of LPO in young patients (up to 30 years) with a disease standing of up to 5 years are significantly higher than in age-matched controls whereas with increasing duration of the disease the levels of this parameter decrease and in elderly patients (over 50 years) with a longer duration of the disease (over 15 years) this parameter is significantly lower than in age-matched controls. A decrease of LPO is particularly pronounced in a bipolar course of manic depressive psychosis.

[Study of the processes of lipid peroxidation in patients with endogenous psychoses using gas chromatography]. / Izuchenie protsessov perekisnogo okisleniia lipidov u bol'nykh éndogennymi psikhozami s pomoshch'iu gazovoi khromatografii.

The processes of lipid peroxidation were studied in healthy subjects and patients with manic-depressive psychosis and schizophrenia using a non-invasive method based on chromatographic determination of endogenic pentane in the expired air. In patients with circular psychosis pentane concentration in the expired air did not differ from the norm, yet with advancing age it decreased (in contrast to an elevation in normal subjects). In schizophrenic patients pentane levels were significantly higher than in age-matched normal subjects. The level of the studied parameter was not correlated with the duration of the disease but showed a definite correlation with the severity of adverse disturbances in schizophrenia.

[Lipid peroxidation processes in patients with schizophrenia]. / Dinamika protsessov perekisnogo okisleniia lipidov v organizme bo'lnykh shizofreniei.

In schizophrenic patients, the course of lipid peroxidation (LP) was investigated as indicated by pentane contents in the expired air (gas chromatography) during a course of psychotropic drug therapy. In 80% of the patients LP was activated before the therapy and reduced in its course with the levels in remission comparable to those characteristic of healthy controls. The LP decreased in a "leap-like" manner with episodes far exceeding the control levels. These leaps' rate and the baseline LP increase before therapy positively correlated with severity of the signs of the disease, its duration, and pre-existing level of negative disorders.

Phenol-oxidizing laccases from the termite gut.

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

[Osmotic resistance and the adhesion capacity of human peripheral blood lymphocytes normally and in schizophrenia]. / Osmoticheskaia rezistentnost' i sposobnost' k adgezii limfotsitov perifericheskoi krovi cheloveka v norme i pri shizofrenii.

A study of resistance to osmotic shock of peripheral blood lymphocytes from healthy donors and schizophrenics demonstrated two lymphocyte subpopulations: a low resistant subpopulation (about 20%) and high resistant subpopulation (about 80%). The lymphocytes from donors belonging to the former subpopulation popossess increased adhesiveness. 60% of schizophrenic patients lack the subpopulation of low resistant lymphocytes. Abnormal resistance to osmotic shock is characteristic of highly adhesive lymphocytes.

Aminopeptidase-like activities in Caenorhabditis elegans and the soybean cyst nematode, Heterodera glycines.

Aminopeptidase-like activities in crude whole body extracts of the free-living nematode Caenorhabditis elegans and the plant parasitic soybean cyst nematode Heterodera glycines were examined. General characteristics including pH optima, heat lability, and inactivation of enzyme by organic solvent were the same for the two species. All developmental stages of H. glycines exhibited activity. In older females, activity was present primarily in the eggs. Affinity for the substrate L-alanine-4-nitroanilide was the same regardless of the stage examined, and was similar for the two species (m for C. elegans and m for H. glycines). Nearly all (>95%) of C. elegans aminopeptidase-like activity was present in the soluble fraction of the extract, while H. glycines activity was distributed between the soluble and membrane fractions. Specific activities of the soluble enzymes were highest in C. elegans and H. glycines juveniles. The C. elegans enzyme was susceptible to a number of aminopeptidase inhibitors, particularly to amastatin and leuhistin, each of which inhibited aminopeptidase-like activity more than 90% at 90 microm. In H. glycines, aminopeptidase-like activity was inhibited 39% by amastatin at 900 microm. The apparent molecular weight of the soluble C. elegans enzyme is 70-80 kDa. Some activity in H. glycines is present in the 70-80 kDa range, but most activity (80-90%) is associated with a very high molecular weight (>240 kDa) component.

Two affinity states of N-methyl-D-aspartate recognition sites: modulation by cations.

Previous studies have indicated that inorganic and organic cations can markedly affect parameters of the function of the N-methyl-D-aspartate receptor ionophore complex. As these effects may involve modulation of agonist binding, the purpose of our study was to investigate the stimulatory effect of mono- and divalent cations on binding properties of glutamate/N-methyl-D-aspartate recognition sites on the N-methyl-D-aspartate receptor complex, using [3H]CGP 39653 as the specific ligand for these sites. In well-washed membranes from rat brain, [3H]CGP 39653 binding sites were present at two affinity states when assayed at 10 mM HEPES-KOH buffer. About 75% of these sites were in a low-affinity state (Kd = 210 +/- 30 nM) although 25% were in a high-affinity state (Kd = 6.4 +/- 0.4 nM). Addition of mono- or divalent cations to the incubation medium stimulated [3H]CGP 39653 binding, measured at a radioligand concentration of 4 nM. Maximal increases in binding were to approximately 230 and 400% of control, in the presence of mono- and divalent cations, respectively. Values of EC50 for stimulation were 5 to 7 mM for monovalent cations and 0.2 to 0.4 mM for divalent cations. At these concentrations, cations increased the Bmax for the high-affinity population of [3H]CGP 39653 sites and decreased the Bmax for low-affinity ones. These findings suggest that, like spermidine, inorganic cations stimulate binding by converting [3H]CGP 39653 binding sites from the low- to high-affinity state.

[Isolation of substances inhibiting the specific binding of imipramine and serotonin uptake from the brain of the bull]. / Vydelenie veshchestv, ingibiruiushchikhh spetsificheskoe sviazyvanie imipramina i zakhvat serotonina, iz golovnogo mozga byka.

Several fractions that inhibit specific binding of 3H-imipramine and reverse uptake of 3H-serotonin in rat brain synaptosomes were obtained by gel chromatography on Sephadex G-10 from an acidic extract of brain homogenates. The profiles of inhibition of 3H-imipramine specific binding and reverse uptake of 3H-serotonin were found to be in a good agreement. This finding suggests that substances identified in the brain extract may have both types of activities. Further investigations relative to the purification and identification of the substances isolated from bovine brain should be aimed at the search for endogenous ligands for the "imipramine receptor".

[Effect of antioxidants on the release of 3H-serotonin by rat brain synaptosomes]. / Deistvie antioksidantov na vybros 3H-serotonina sinaptosomami mozga krys.

The effects of free radical scavengers of different chemical structure (hydroxypyridines, hindered phenols, hydroxyphenothiazines) on processes of 3H-hydroxytryptamine uptake and release by rat brain synaptosomes were studied. All free radical scavengers used inhibited 3T-hydroxytryptamine uptake and stimulated 3H-hydroxytryptamine release, with the efficacy being reduced in the following order: 7-hydroxyaminazine greater than butylated hydroxytoluene greater than paginol-2-methyl-6-ethyl-3-hydroxypyridine greater than alpha-tocopherol. The effects of free radical scavengers were eliminated in the presence of EGTA or after cooling to 4 degrees C.

[Antioxidant-induced release of calcium in biological membranes]. / Indutsiruemye antioksidantami utechki kal'tsiia v biologicheskikh membranakh.

The effect of synthetic (BHT, 2,2,5,7,8-pentamethyl-6-hydroxychroman) and natural (alpha-tocopherol) antioxidants on Ca++-transporting systems was compared in platelets, brain synaptosomes, and skeletal muscle sarcoplasmic reticulum. It was shown that synthetic antioxidants, in contrast to alpha-tocopherol, induced Ca++-release manifested in platelet aggregation, stimulation of 5-hydroxytryptamine release by synaptosomes, synaptosome depolarization and inhibition of Ca++-transport and Ca++-ATPase activity in the sarcoplasmic reticulum. The disturbances of Ca++-homeostasis induced by synthetic antioxidants are considered as molecular mechanisms of complications encountered upon their application.

[Thrombocytic serotoninergic markers in therapy-resistant patients with endogenous depression undergoing alpha-tocopherol treatment]. / Trombotsitarnye serotoninergicheskie markery u rezistentnykh k terapii bol'nykh éndogennymi depressiiami pri lechenii al'fa-tokoferolom.

The main platelet serotoninergic parameters (maximal velosity of 3H-serotonin uptake, Vmax and density of 3H-imipramine binding sites, Bmax) were measured in 17 therapy-resistant endogenously depressed patients before and after combined treatment (antidepressants and alpha-tocopherol). It was found a significant decrease of Vmax-, Vmax/Bmax- values and the increase of Bmax- values in patients before treatment. A beneficial effect of the combined therapy was found in 47% of patients. However, it was not followed by the changes of main platelet serotoninergic parameters in patients after treatment in spite of the obtained clinical improvement. There were not also observed the significant differences of measured biochemical parameters between responders and non-responders. These findings suggest that the beneficial clinical effect of the combined antidepressive therapy with alpha-tocopherol does not involve serotoninergic mechanisms.