Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

Zinc protects against arsenic-induced apoptosis in a neuronal cell line,measured by DEVD-caspase activity

Acute and chronic arsenic exposure results in toxicity in humans and causes many neurological and other manifestations. For the first time the present study reports that zinc decreases arsenic-induced apoptosis and also confirms a single report of apoptosis induced by arsenic in a neuronal cell line. Apoptosis measured by DEVD-caspase activity peaked between 10 M and 20 M of arsenic trioxide. Higher concentrations of arsenic up to 40 M caused increasing cell death with diminishing DEVD-caspase activity. The beneficial effect of zinc was proportional to its concentration with a significant decrease in arsenic-induced DEVD-caspase activity at 50 M and 75 M zinc (P<0.05). This finding may be of therapeutic benefit in people suffering from chronic exposure to arsenic from natural sources, a global problem especially relevant to millions of people on the Indian subcontinent.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Polyamines and the development of isolated neurons in cell culture

The possible role of polyamines in the development of isolated neuroblasts from the cerebral cortex of embryonic chick brain was studied by means of three enzyme activated irreversible inhibitors of ornithine decarboxylase. -Difluoromethylornithine (MDL 71782) showed no effects on development at doses which depleted dramatically neuronal putrescine and spermidine levels. In contrast, the two other inhibitors, (E)--(fluoromethy)dehydroputrescine (MDL 72197) and 6-heptyne-2,5-diamine (MDL 72175) blocked the formation of neuronal outgrowths completely at 100 M and higher concentrations. Their effects on neuronal polyamines differed at this concentration considerably. The growth inhibitory effect of the ornithine decarboxylase inhibitors was in all cases reversible: cells which were grown after 3 days of exposure to the drugs in normal medium produced neuronal networks. The presence of putrescine at 10M concentration in the culture medium prevented the growth inhibitory effect of 100 M concentrations of the drugs. This concentration of putrescine was not only capable of preventing, but also of reversing growth inhibition by the ornithine decarboxylase inhibitors. Although the cellular polyamine levels were not correlated with the morphological development of chick embryo cortical neurons, the present study leaves no doubt that putrescine plays an essential role in neuronal differentiation.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Isolation and culture of Lens culinaris Medik. cv. Eston epicotyl protoplasts to calli

Protoplasts of Lens culinaris Medik. cv. Eston were isolated from epicotyl tissues of seedlings grown on Murashige & Skoog basal medium. For isolating the protoplasts, epicotyl tissues were digested for 12–14 h at 25°C in an isolation mixture (pH 5.7) containing 1% Cellulase RS, 0.5% Driselase, 0.25% Pectolyase Y23, 0.2M calcium chloride, 10 mM mannitol and 10 mM MES. Protoplasts were purified by flotation over 20% sucrose and washed with 0.2 M calcium chloride solution supplemented with 10 mM mannitol. Purified protoplasts were cultured at a density of 10⁵ ml^-1 in agarose (Seaplaque, 0.6%) blocks which were suspended in an identical but liquid KM8P culture medium lacking amino acids, ammonium nitrate, and coconut water but containing 0.35 M glucose and a growth regulator complement of either 2.2 M 2,4-dichlorophenoxyacetic acid (2,4-D), 2.7 M naphthaleneacetic acid (NAA), 2.3 M N-(2-furanylmethyl)-1H-purine-6-amine (kinetin), 2.2 M benzylamino purine (BAP), 2.3 M 2-methyl-4-(1H-purine-6-ylamino)-2-buten-1-ol (zeatin), and 1.4 M gibberellic acid (GA₃), or 5.4 M NAA and 2.2 M each of 2,4-D and BAP. The osmotic potential of the liquid culture medium was gradually reduced over a period of 3 weeks by replacing the spent medium with a fresh medium containing 0.25, 0.1 and 0 M glucose at weekly intervals. About 6% of the dividing protoplasts developed into cell colonies after 3 weeks of culture at 25°C in diffuse light (10 E m^-2s^-1). In 35–42 days the microcolonies were about 1 mm in diameter and developed into calli on transfer to agar-solidified B5 medium supplemented with growth regulators used in the protoplast culture medium and 5 mM glutamine. Attempts to regenerate plants from protoplast-derived calli have so far been unsuccessful.Department of Applied Microbiology and Food Science, University of Saskatchewan     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Polyamines and proline are affected by cadmium stress in nodules and roots of soybean plants

The effect of moderate (50 M) and high (200 M) doses of Cd were studied in relation to polyamine (Pas) metabolism, proline level and the glutamine synthetase/glutamate synthase system (GS/GOGAT) activity in nodules and roots of soybean plants during 6 days of treatment. The lower Cd concentration increased putrescine (Put) in both nodules and roots, while 200 M Cd increased Spm only in nodules and Put in roots. Spermidine (Spd) decreased in roots under both Cd concentrations. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were both involved in Put biosynthesis in roots. In nodules, Put formation could mainly be attributed to ODC activity. Diamine oxidase (DAO) activity was severely reduced by 50 and 200 M Cd either in nodules or roots. The GS/GOGAT system activity was depressed either with 50 or 200 M Cd, but most significantly with the highest metal concentration. Under 200 M Cd, GS activity decayed to 25% or 60% of the control in nodules and roots, respectively, while GOGAT decreased 85% in nodules and 79% in roots by day 4 of treatment. Ammonium increased greatly in nodules (200% over the controls) and roots (100%) under 200 M Cd. Proline concentration increased significantly in nodules and roots under both Cd treatments, more markedly under 200 M Cd. The relationship between Pas and proline accumulation and nitrogen assimilation is discussed.     

Influence of sarmesin on some dopamine-related types of behaviour

Summary The present study was undertaken to investigate the effects of sarmesin, an analogue of [Sar¹] angiotensin II (ANG II) where the tyrosine hydroxyl group in position 4 is methylated, on dopamine (DA)-related paradigms: locomotor and exploratory behaviour as well as apomorphine (3 mg/kg, ip)-induced stereotypy in rats. Sarmesin (0.5 and 1 g, icv) significantly decreased ambulation and rearing movements, and blocked the inhibitory effect of ANG II (0.1 g) on both types of activity. Sarmesin induced biphasic effects on apomorphine-induced stereotypy depending on the dose increase (0.5 and 5 g, icv) and decrease (10 g). Moreover, sarmesin (5 g) blocked the inhibitory effect of ANG II (2 g, icv) on apomorphine stereotypy. Taken together, these results suggest that sarmesin might interact with AT₁ and AT₂ receptor subtypes. The results further confirm the statement for ANG II-DA interaction in brain structures involved in these types of behaviour.     

Calcein as a fluorescent iron chemosensor for the determination of low molecular weight iron in biological fluids

The fluorescence quenching of calcein (CA) is not iron specific and results in a negative calibration curve. In the present study, deferoxamine (DFO), a strong iron chelator, was used to regenerate the fluorescence quenched by iron. Therefore, the differences in fluorescence reading of the same sample with or without addition of DFO are positively and specifically proportional to the amounts of iron. We found that the same iron species but different anions (e.g. ferric sulfate or ferric citrate) differed in CA fluorescence quenching, so did the same anions but different iron (e.g. ferrous or ferric sulfates). Excessive amounts of citrate competed with CA for iron and citrate could be removed by barium precipitation. After optimizing the experimental conditions, the sensitivity of the fluorescent CA assay is 0.02 M of iron, at least 10 times more sensitive than the colorimetric assays. Sera from 6 healthy subjects were tested for low molecular weight (LMW) chelator bound iron in the filtrates of 10 kDa nominal molecular weight limit (NMWL). The LMW iron was marginally detectable in the normal sera. However, increased levels of LMW iron were obtained at higher transferrin (Tf) saturation (1.64–2.54 M range at 80% Tf saturation, 2.77–3.15 M range at 100% Tf saturation and 3.09–3.39 M range at 120% Tf saturation). The application of the assay was further demonstrated in the filtrates of human liver HepG2 and human lung epithelial A549 cells treated with iron or iron-containing dusts.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Corticosteroids inhibit the generation of lymphokine-activated killer activity in vitro

Summary In phase-I clinical trials of adoptive immunotherapy using lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) (Cetus) for the treatment of malignant glioma, we observed that blood mononuclear cells (MNC) from patients dependent on dexamethasone for management of cerebral edema produced substantially less LAK activity as compared to MNC of normal blood donors or glioma patients not receiving steroid therapy. Therefore, we examined the in vitro effects, brought about by therapeutically attainable concentrations of various corticosteroids, on the proliferative response, production of interferon (IFN-), and induction of LAK activity from blood MNC of normal donors. Incubation in media containing rIL-2 (1000 U/ml) with either dexamethasone, hydrocortisone, methylprednisolone, or prednisolone profoundly affected all of these parameters. First, while 0.01 g/ml of either dexamethasone or hydrocortisone caused a slight enhancement of the mitogenic response of lymphocytes to phytohemagglutinin, a dose-dependent decline occurred as concentrations increased to 10 g/ml. The addition of prednisolone and methylprednisolone elicited a dose-dependent inhibition of lymphocyte proliferation over the entire concentration range tested. At 0.1 g/ml or higher, dexamethasone, hydrocortisone, methylprednisolone and prednisolone significantly (P<0.02) inhibited the production of IFN-: respectively 18.9%, 4.4%, 2.2%, and 12.3% of the IFN- produced by MNC in the absence of steroids. All four corticosteroids inhibited the induction of LAK activity. Compared to MNC that had been incubated with 1000 U/ml rIL-2 alone, MNC cultured with rIL-2 and 10 g/ml either dexamethasone or prednisolone demonstrated significantly lower cytotoxicity (P<0.05) for the natural-killer-cell-resistant cell line, Daudi. Culturing MNC with hydrocortisone had a more dramatic result, causing a significant decline (P<0.01) in lytic activity at both 1.0 g/ml and 10 g/ml, while incubation with methylprednisolone produced a significant drop (P<0.02) in LAK-mediated cytotoxicity at 0.1 g/ml as well as 1.0 g/ml and 10 g/ml. When cytotoxicity was expressed as lytic units per million effectors, a dose-response decline in lytic activity was once again apparent, with hydrocortisone, methylprednisolone and prednisolone showing significant inhibition (P<0.05) at both 1.0 g/ml and 10 g/ml and dexamethasone at 10 g/ml (P<0.01). These results indicate that corticosteroids commonly used in the management of cerebral tumors and other malignancies inhibit induction of LAK activity in vitro, and this may explain why it is often difficult to generate LAK activity from blood MNC of patients who are receiving chronic steroid therapy.     

The selective inhibition of hippocampal glutamic acid decarboxylase in zinc-induced epileptic seizures

The intracerebroventricular administration of Zn²⁺ (0.3 mol/10 l) causes epileptic seizures characterized by running fits, jumping, vocalization, fasiculation of facial muscles, myoclonic movements of the limbs and tonic-clonic convulsions. These episodes are blocked or reversed by -aminobutyric acid (0.4 mol/10 l). When assayed under conditions where pyridoxal phosphate was not added, the activity of glutamic acid decarboxylase decreased significantly in hippocampus from 18.9 to 15.3 and 9.7 mol¹⁴CO₂ formed/gram proteins/20 min, 15 and 30 min following administration of Zn²⁺. The inhibition of glutamic acid decarboxylase by Zn²⁺ was selective occurring only in hippocampus and not in the hypothalamus, amygdala, caudate or thalamus. The inhibition of glutamic acid decarboxylase was not due to a reduction in the concentration of endogenous pyridoxal phosphate which remained unaltered in hippocampus following Zn²⁺ administration.     

In vitro meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

Isolation and culture of protoplasts from young sporophytes ofSalvinia natans aseptically obtained by co-culture of female and male gametophytes

Sporophytes were aseptically obtained by co-culture of female and male gametophytes derived from two types of spores (megaspores and microspores) of the heterosporous fernSalvinia natans All. Protoplasts isolated enzymatically from juvenile leaflets of sporophytes were cultured in a 1/10 Murashige and Skoog's medium containing 2.2 M naphthalene acetic acid, 2.2 M 6-benzyl-aminopurine, 0.35 M mannitol, and 0.05 M sucrose. Cell division took place within 6 days of culture, and cell-clusters composed of 9–10 cells were observed after 30 days of culture.Abbreviations BA 6-benzyl-aminopurine - MS Murashige and Skoog - NAA naphthaleneacetic acid     

A novel technique for the study of bacterial cell mechanical properties

A micromanipulation method is described for measuring the bursting forces of bacteria and relating them to cell size. At a compression speed of 6.2 m s^–1, bursting forces of three samples of rapidly growing Staphylococcus epidermis from a batch culture varied from 3 to 34 N with an average value of 13.8 N (standard error 0.8 N). Escherichia coli grown in continuous culture at a specific growth rate of 0.5 h^–1 had bursting forces varying from 1 to 9 N with an average value of 3.6 N (standard error 0.4 N). In squeeze-hold experiments, force relaxation was observed, which was attributed to water loss from the cells, or viscoelasticity, or both. At high compression speed, such as 6.2 m s^–1, this relaxation could be neglected. Micromanipulation strength measurements might be used in studies of cell mechanical disruption and of the dependence of cell strength on cell physiology.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

In vitro propagation of Glehnia littoralis from shoot-tips

Shoot cultures of Glehnia littoralis F. Schmidt ex Miq. (Umbelliferae) were established by placing shoot tip explants on Linsmaier and Skoog medium with 1 M NAA and 10 M BAP. Shoots were multiplied on the basal medium supplemented with 0.3 M NAA and 3 M BAP and rooted on medium containing either 1 M IBA or 3–10 M IAA. Plantlets survived in pots without any covering. This unique characteristic of the plantlets was ascribed partly to a well-developed cuticle on the surface of the leaf and the small ratio of surface area to fresh weight of a leaf blade in comparison with those of other species whose plantlets needed coverings after potting. The regenerated plantlets were finally transferred to soil.Abbreviations IAA potassium indole-3-acetate - IBA indole-3-butyric acid - IPA indole-3-propionic acid - NAA potassium 1-naphthaleneacetate - 2,4-D sodium 2,4-dichlorophenoxyacetate - BAP 6-benzylaminopurine - 2-iP N⁶-(2-isopentenyl)adenine     

Regional and subcellular distribution of taurine-synthesizing enzymes in the rat central nervous system

The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94–99 mol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44–51 mol/h/g; cerebellum, 71 mol/h/g; pons-medula and spinal cord, 94 and 60 mol/h/g, respectively. The distribution of CSD activity expressed as mol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14–21 mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8–13 mol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, colliculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.     

Regeneration of pea (Pisum sativum L. cv. Century) plants by in vitro culture of immature leaflets

In vitro regeneration of plants from immature leaflets of 3 day-old pea (Pisum sativum L. cv. Century) seedlings was studied under defined nutritional, hormonal and environmental conditions. Immature leaflets isolated from the second and third apical leaves of aseptically germinated seeds were cultured on MS medium containing vitamins as in B5 medium, 3% sucrose, 0.8% agar and supplemented with 0.1, 1, and 10 M concentrations of naphthaleneacetic acid (NAA) and 1 and 10 M levels of benzyladenine (BA) in various combinations. Shoot regeneration from the primary callus occurred within 45 to 90 days of culture in most of the hormone combinations. Although the number of calli producing shoots was maximal at 10 M levels of NAA and BA, multiple shoot regeneration was predominant at a combination of 0.1 M NAA and 10 M BA. Indoleacetic acid (IAA) and kinetin (K), both at 10 M, also induced shoot regeneration. No shoots were regenerated when 10 day-old leaflets were used as explants. Root production generally occurred on non-shoot regenerating calli. Roots were induced to differentiate by transferring the regenerated shoots onto half-strength B5 medium supplemented with 1 M NAA.NRCC # 19712Visiting Scientist, supported by a research fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (Rep. Argentina). Permanent address: Facultad de Ciencias Agrarias, IBONE, Casilla de Correos 209, Corrientes (3400), Argentina.