Ambivalent effects of defective DNA in beet curly top virus-infected transgenic sugarbeet plants.

Beet curly top virus (BCTV) limits sugarbeet production considerably. Previous studies have shown that infections are associated with the generation of defective DNAs (D-DNA) which may attenuate symptoms. Transgenic sugarbeet lines were established carrying a partial direct repeat construct of D-DNA in order to examine whether they are useful as a means of generating tolerance against BCTV. Thirty four independent transgenic lines were challenged. Viral full-length and D-DNAs were monitored by polymerase chain reaction (PCR) or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP). The differential accumulation of both DNA species was compared with symptom severity during the course of infection. RCA/RFLP allowed the discrimination of two D-DNA classes which were either derived from the transgenic construct (D(0)) or had been generated de novo (D(n)). The statistical analysis of the results showed that the presence of D(0)-DNA correlated with increased symptom severity, whereas D(n)-DNAs correlated with attenuated symptoms.

A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions.

Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB(-)) and [W(2)(CO)(10)(S(2)CH)](-), the putative lipophilic anion B(CF(3))(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB(-) and B(CF(3))(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB(-) and B(CF(3))(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.

Homocystinuria due to cystathionine beta-synthase deficiency: novel biochemical findings and treatment efficacy.

To explore the pathogenesis of cystathionine beta-synthase (CBS) deficiency and to test the efficacy of pharmacological therapy we examined a panel of metabolites in nine homocystinuric patients under treated and/or untreated conditions. Off pharmacological treatment, the biochemical phenotype was characterized by accumulation of plasma total homocysteine (median 135 micromol/L) and blood S -adenosylhomocysteine (median 246 nmol/L), and by normal levels of guanidinoacetate and creatine. In addition, enhanced remethylation was demonstrated by low serine level (median 81 micromol/L), and by increased concentration of methionine (median 76 micromol/L) and N -methylglycine (median 6.8 micromol/L). Despite the substantially blocked transsulphuration, which was evidenced by undetectable cystathionine and severely decreased total cysteine levels (median 102 micromol/L), blood glutathione was surprisingly not depleted (median 1155 micromol/L). In 5 patients in whom pharmacological treatment was withdrawn, the differences of median plasma total homocysteine levels (125 micromol/L after withdrawal versus 33 micromol/L under treatment conditions), total cysteine levels (139 versus 211 micromol/L) and plasma serine levels (53 versus 103 micromol/L) on and off treatment demonstrated the efficacy of long-term pyridoxine/betaine administration ( p <0.05). The treatment also decreased blood S -adenosylhomocysteine level (133 versus 59 nmol/L) with a borderline significance. In summary,our study shows that conventional treatment of CBS deficiency by diet and pyridoxine/betaine normalizes many but not all metabolic abnormalities associated with CBS deficiency. We propose that the finding of low plasma serine concentration in untreated CBS-deficient patients merits further exploration since supplementation with serine might be a novel and safe component of treatment of homocystinuria.

Transsulfuration in Saccharomyces cerevisiae is not dependent on heme: purification and characterization of recombinant yeast cystathionine beta-synthase.

Cystathionine beta-synthase [CBS; L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration in both yeast and humans. It has been established previously that human CBS is a hemeprotein but although the heme group appears to be essential for CBS activity, the exact function of the heme group is unknown. CBS activity is absent in heme deficient strains of Saccharomyces cerevisiae grown without heme supplementation. CBS activity can be restored by supplementing these strains with heme, implying that there is a heme requirement for yeast CBS. We subcloned, overexpressed and purified yeast CBS. The yeast enzyme shows absolute pyridoxal 5'-phosphate (PLP) dependence for activity but we could find no evidence for the presence of a heme group. Given the degree of sequence and mechanistic similarity between yeast and human CBS, this result indicates that heme is unlikely to play a direct catalytic role in the human CBS reaction mechanism. Further characterization revealed that, in contrast to human CBS, S-adenosylmethionine (AdoMet) does not activate yeast CBS. Yeast CBS was found to be coordinately regulated with proliferation in S. cerevisiae. This finding is the most likely explanation of the observed apparent heme dependence of transsulfuration in vivo.

Intraoperative test stimulation with a modified implantable pulse generator in deep brain stimulation.

A modified implantable pulse generator (MIPG) for intraoperative test stimulation in chronic deep brain stimulation is described. The MIPG can be used for bipolar stimulation with quadripolar electrodes. The device is programmed and controlled with a standard console programmer. It can also be used for postoperative test stimulation with externalized electrodes. In our experience, the MIPG has several advantages as compared to the screener that is usually used.

Synthesis and anti-HIV activity of alpha-thiophenoxy-hydroxyethylamide derivatives.

A series of new anti-HIV derivatives containing a novel alpha-thiophenoxyhydroxyethylamide core have been synthesized, using S-phenylbenzenethiosulfonate as the thiosulfenylating reagent. Some of the new synthesized compounds (1a, 1c, 1g, 1i, 1j and 1l) inhibited HIV replication in cell culture assays (syncytia formation) with effective concentrations (EC(50)) ranging from 0.1-1 microM. Incorporation of thiophenoxy substitution within various pseudomimetic peptide backbones provided a series of highly potent HIV inhibitors.

Astra blue and basic fuchsin double staining of plant materials.

Methods for double staining plant materials using astra blue and basic fuchsin are described here. These methods can be applied to free hand and microtome sections embedded in paraffin, paraplast or historesin. Also, they can be used to study isolated epidermal peels and pollen preparations. Temporary, semipermanent and permanent preparations were studied. Astra blue stained polysaccharides of the cell wall such as cellulose and pectins. Basic fuchsin showed an affinity for lignified, suberized or cutinized walls. The easy preparation of the reagents, excellent color contrast of the histological preparations, and brief staining times of some methods makes them useful for both routine research and didactic purposes. Also, excellent color or black and white photomicrography can be obtained after the double staining described here.

Complete spontaneous remission in a patient with metastatic non-small-cell lung cancer. Case report, review of the literature, and discussion of possible biological pathways involved.

Spontaneous remission of cancer (SR) is defined as a complete or partial, temporary or permanent disappearance of all or at least some relevant parameters of a soundly diagnosed malignant disease without any medical treatment or with treatment that is considered inadequate to produce the resulting regression. We report the case of a 61-year-old man who presented with extensive metatastic disease five months after pneumonectomy for poorly differentiated large cell and polymorphic lung cancer. A vast metastatic tumour mass of the abdominal wall was confirmed histolologically and there was clinical and radiographic evidence of liver and lung metastases. Eight months later, the patient was operated on for a hernia, which had developed in the inguinal biopsy scar and the surgeon confirmed complete clinical SR of the abdominal wall metastases. Again five months later there was no longer any radiologic evidence of liver and lung metastases. Complete remission has persisted more than five years. Histology of the primary and of the abdominal metastases were reviewed by several independent pathologists. SR is an extremly rare event in lung cancer. This is the first documented case of clinically evident visceral metastases of a bronchiogenic adenocarcinoma developing after complete resection of the primary and then showing complete SR. The epidemiology of SR is reviewed and possible mechanisms involved in SR are discussed.

The tRNATyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron-containing pre-tRNAs in wheat germ extract.

Southern analysis of Triticum DNA has revealed that nuclear tRNATyr genes are dispersed at a minimum of 16 loci in the genome. We have isolated six independent tRNATyr genes from a Triticum aestivum library in addition to three known members of the Triticum tRNATyr family. Four of the sequenced tRNATyr genes code for Triticum tRNA Tyr and two code for tRNA2Tyr. Three genes encode tRNAsTyr which carry one or two nucleotide substitutions as compared to the conventional genes. The nine Triticum tRNATyr genes possess highly conserved intron sequences ranging in size from 12 to 14 nucleotides. A common secondary intron structure with the 5' and 3' splice site loops separated by five base pairs can be formed by all pre-tRNAs Tyr which are efficiently spliced in the homologous wheat germ extract.

Hyperhomocysteinemia in premature arterial disease: examination of cystathionine beta-synthase alleles at the molecular level.

Hyperhomocysteinemia occurs in approximately 30% of the patients with premature occlusive arterial disease (POAD). Some of these exhibit significantly reduced fibroblast cystathionine beta-synthase (CBS) activities, suggesting that they may be heterozygous for CBS deficiency. To test this possibility, we studied cDNA derived from four well characterized patients with POAD, exhibiting hyperhomocysteinemia and reduced CBS activities, from four normal controls, and from four obligatory heterozygotes for CBS deficiency. Lysates of individual colonies of E.coli, containing full-length PCR-amplification products in the expression vector, pKK388.1, were tested for CBS activity. cDNA from at least seven of the eight possible independent POAD alleles encoded catalytically active, stable CBS which exhibited normal response to both PLP and AdoMet. The sequences of all 3'-untranslated regions of all seven isolated POAD alleles were identical to the normal, 'wild-type' CBS sequences. The results of the expression studies were confirmed for one POAD patient by determining the full-length cDNA sequences for both alleles; these were entirely normal over the complete length of the cDNA. In contrast, the screening method correctly distinguished mutant from normal alleles in all four obligatory heterozygotes studied. We conclude that CBS mRNAs from POAD individuals are free from inactivating mutations, including all 33 previously identified in heterozygous carriers and homocystinuric patients.

Human cystathionine beta-synthase cDNA: sequence, alternative splicing and expression in cultured cells.

Cystathionine beta-synthase (CBS) deficiency is the major cause of homocystinuria in humans. The most frequent symptoms of homocystinuria include: dislocated optic lenses, vascular disorders, skeletal abnormalities and mental retardation. Patients with this deficiency have elevated levels of homocyst(e)ine, methionine and low cysteine in their body fluids. These abnormal levels often partially or fully normalize upon treatment with pharmacological doses of vitamin B6. To investigate the molecular and biochemical basis for these conditions, it was necessary to determine the nucleotide and polypeptide sequence of CBS. We report here the human CBS cDNA sequence of 2,554 nucleotides encoding the CBS subunit of 551 amino acids. An intron of 214 bp appears to be retained in the 3'-untranslated region of most of the fibroblast and liver mRNA. We also report a frequent Mspl polymorphism in the 3'-untranslated sequence and two synonymous mutations in the coding region: 699C/T (Y233Y) and 1080C/T (A360A). The amino acid sequence similarity of human and rat CBS is greater than 90%; the enzyme also exhibits 52% similarity to O-acetylserine(thiol)-lyase from bacteria and plants. Lastly, we demonstrate that expression of the human enzyme in CHO cells yields enzymatically active protein of the expected size with a half-life of approximately 14 hrs.

[Regional cytostatic perfusion of the extremities in patients with malignant melanoma and soft tissue sarcoma--therapeutic applications and results]. / Die regionale zytostatische Extremitätenperfusion bei Patienten mit malignem Melanom und Weichteilsarkom--therapeutische Anwendungen und Ergebnisse.

94 patients with malignant melanoma or soft tissue sarcoma of the extremities were submitted to isolated cytostatic limb perfusion. With palliative indication for treatment of malignant melanoma, the 5 year survival rate was 25% after perfusion with methotrexate and 50% after perfusion with melphalan. The adjuvant perfusion showed very good results, too. Furthermore, the authors report on their first experiences in regional cytostatic perfusion of soft tissue sarcoma and bone sarcoma.

Antitumor activity of cell wall beta-1,3/1,6-glucans from Phytophthora species.

From the cell walls of various Phytophthora species (Oomycetes) different water-soluble beta-1,3/1,6-glucans were isolated and characterized. The glucans with relative molecular masses between 13 and 50 kd exhibited varying degrees of branching in the beta-1,3-linked glucan backbone ranging from 14 to 50%. The dose-dependent antitumor activity of the glucans against allogeneic sarcoma-180 was investigated. It could be demonstrated that beta-1,3/1,6-glucans with relative molecular masses below 50 kd can exhibit a prominent antitumor activity (inhibition rate up to 99%), being comparable to that of the high relative molecular mass (450 kd) beta-1,3/1,6-glucans. Biological activity of the glucans could be shown to be correlated with a low degree of branching. The most potent glucan with a degree of branching of 14% also was active against the syngeneic DBA/2-MC.SC-1 fibrosarcoma (inhibition rate up to 90%) and in combination with a suboptimum dose of diethylstilbestrol against Nobel-Nb prostate carcinoma (inhibition rate up to 90%).

[Polysaccharides from Nerium oleander: structure and biological activity]. / Polysaccharide aus Nerium oleander: Struktur und biologische Aktivität.

A water extraction of crushed leaves of Nerium oleander yielded 2.3% of a crude polysaccharide. The main fraction (67%) represents a pectic polysaccharide mainly composed of galacturonic acid besides rhamnose, arabinose and galactose. The polysaccharide structure was characterized by NMR, mild acid- and pectinase treatment combined with GC-MS analyses. In vivo tests for a possible antitumor activity did not result in a significant action. Investigation of immunomodulating activity brought some indications for mitogenic activity and a weak macrophage-mediated cytotoxicity. Increase of phagocytosis could not be doubtlessly assigned to the polysaccharide due to the high amount of endotoxin in the homogenous fraction OLE 2.

Chemical structure and biological activity of water-soluble polysaccharides from Cassia angustifolia leaves.

The water-soluble polysaccharides from Cassia angustifolia L. leaves were isolated and fractionated. The acidic polysaccharide fraction was separated into two subfractions S1 and S2 consisting of L-rhamnose, L-arabinose, D-galactose, and D-galacturonic acid. Further fractionation of the predominant S1 by GPC gave two fractions S1A and S1B with an average molecular weight of 2 x 10(6) and 1.5 x 10(5) d, respectively. Methylation analysis of S1A showed the presence of 1,4-linked galacturonic acid (31.0%), 1,2-linked rhamnose (14.5%), 1,2,4-linked rhamnose (15.8%), 1,3,6-linked galactose (15.3%), smaller amounts of 1,3-linked arabinose, 1.5-linked arabinose, and terminal galactose and arabinose residues. Mild acid hydrolysis of S1A indicated that the backbone consists of 1,4-linked galacturonic acid and 1,2-linked rhamnose residues in the ratio of 1:1. Every second rhamnose is connected via C-4 to arabinogalactan sidechains. The antitumor activity of the polysaccharide fractions was tested against the solid Sarcoma-180 in CD1 mice. Only S1A exhibited a significant antitumor activity with an inhibition rate of 51%.

Action de differentes hauteurs de dilution de Phosphorus blanc "Phosphorus" sur la cinetique d'une reaction enzymatique in vitro, impliquant le transfert d'un groupement phosphate / The action of differents of dilution of Phosphorus in thy cinetic of a enzimatic reaction in vitro the transference of one phosphated groupment

On a enregistre des modifications de l'activite enzymatique de la pyruvate kinase, lorsque cette enzyme est incubee dans une milieu contenant outre les cofacteurs ADP et ion Mg++, des dilutions infinitesimales de phosphore blanc "phosphorus"

Acute phase of drug withdrawal management by clothiapine (Entumin): a pilot project.

Fifty-nine drug addicts and alcoholics were treated with clothiapine for a period up to 2 months for alleviation of their withdrawal symptoms. Most of the patients cooperated with the treatment, and this was due to the very few side effects that were experienced by them. The patients were completely withdrawn from physical dependence on drugs and we were able to discharge them without an irresistible impulse for the drug.

Affinity of cystathionine beta-synthase for pyridoxal 5'-phosphate in cultured cells. A mechanism for pyridoxine-responsive homocystinuria.

Previous attempts to correlate in vivo pyridoxine-responsiveness with in vitro assays of cystathionine beta-synthase activity in synthase-deficient homocystinuric patients have been only partially successful. All such studies, however, have been conducted with extracts of cultured skin fibroblasts grown in medium containing a high concentration (1,000 ng/ml) of pyridoxal. Having recently shown that such growth conditions may obscure important aspects of enzyme-coenzyme interactions by saturating most synthase molecules with their cofactor, pyridoxal 5'-phosphate, we have established conditions for growth of cells in pyridoxal-free medium. Under these conditions, intracellular pyridoxal 5'-phosphate fell by >95%, and saturation of cystathionine beta-synthase apoenzyme with pyridoxal 5'-phosphate decreased from a predepletion value of 70% to <10%. When such depleted cells were grown in media containing pyridoxal concentrations ranging from 0 to 1,000 ng/ml, cellular pyridoxal 5'-phosphate reached a maximum of 30 ng/mg cell protein at a medium pyridoxal concentration of 100 ng/ml. Maximal saturation of aposynthase with coenzyme in control cells was reached at a medium pyridoxal concentration of 10 ng/ml. In contrast, maximal saturation of residual aposynthase in cells from an in vivo responsive patient was achieved at a medium pyridoxal concentration of 25-50 ng/ml, whereas that from cells from an in vivo unresponsive patient was reached at 100 ng/ml. Estimates of the affinity of control and mutant cystathionine beta-synthase for pyridoxal 5'-phosphate in cell extracts supported the differences observed in intact cells. The apparent K(m) of cystathionine beta-synthase for pyridoxal 5'-phosphate in extracts of depleted cells from four in vivo-responsive patients was two to four times that of control. In contrast, the K(m) for pyridoxal 5'-phosphate in two lines from in vivo nonresponsive patients was 16- and 63-fold normal. These results suggest that cystathionine beta-synthase activity in cells from patients containing a mutant enzyme with a moderately reduced affinity for pyridoxal 5'-phosphate can be increased by pyridoxine supplements in vivo, whereas that from patients whose enzyme has a more dramatically reduced affinity for the coenzyme cannot be so modulated because of limits on the capacity of such cells to accumulate and retain pyridoxal 5'-phosphate.