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     

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.     

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions

1. The effect of guanosine on L-[2,3-³H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 M) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 M) but not a low (1 M) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 M also increased 100 M glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.     

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.     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Re-organization of microtubules during preprophase band development inAdiantum protonemata

Summary Microtubule organization during preprophase band development was investigated using immunofluorescence microscopy in filamentous protonemal cells (approx. 600 m in length, 20 m in width) ofAdiantum capillus-veneris L. Protonemata pre-cultured under red light were transferred to continuous blue light or total darkness to induce synchronous cell division. Preprophase bands were found under both light conditions. In an early stage of development, the preprophase band which is transverse to the cell axis overlapped with an interphase cortical array of microtubules which is random or parallel to the cell axis. The interphase cortical array disappeared thereafter. While the width of the preprophase band became narrow during development under dark conditions, under blue light conditions it did not.Spatial and temporal aspects of the disappearance of the interphase cortical array of microtubules were also investigated. The interphase cortical array began to disappear at nearly the same time as the beginning of preprophase band formation. Under blue light, the disruption of cortical microtubules started at approx. 150 m from the tip (approx. 120 m from the nucleus), and spread toward the tip as far as the nuclear region and toward the base to an area approx. 300–400 m from the tip. Cortical microtubules remained in the basal part of the protonema. The pattern of disappearance between the tip and nucleus could not be determined. Under dark conditions, the pattern of the disappearance of cortical microtubules was somewhat different in many cells from that encountered with exposure to blue light. Microtubules first re-oriented from longitudinal to transverse, and then gradually disappeared. In some cells, the pattern of disappearance was similar to that observed under blue light.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - ICM interphase cortical microtubules - PBS phosphate buffered saline - PPB preprophase band - MT microtubule     

Morphology and structural organization of spiracles in femaleArgas (Persicargas) walkerae (Acari: Argasidae)

Scanning electron microscopical investigations of fractures and corrosion casts of the spiracles from femaleA. walkerae ticks revealed a four-part structure, consisting of spiracular plate, ostium and macula forming the external closure, followed by the subostial space and the vestibulum of regulable volume, as well as the atrial chamber as the innermost part from which the main tracheal trunks originate. On the average, the spiracular plate was 158 m long and 188 m at the broadest width. It consisted of a thin, highly perforated external and a thick internal layer, which enclosed the interpedicellar space with numerous stout pedicels. In its posterior region, the spiracular plate was covered by the macula, which was up to 80 m in length and 110 m in width. The interpedicellar cavity opened into the subostial space measuring 95.5 m in length and 159.6m in width, which proceeded into the 112-m long vestibulum. The roof of the vestibulum was flexible and could be everted and inverted. Inverted, the roof formed a quadratic bulge with numerous deep cuticular folds, which confined the lumen of the vestibulum either partially or completely. In corrosion casts, the roof was everted to a length of up to 89.3 m. In the posterior part of the vestibulum, as well as in the initial fourth of the artrial chamber, numerous anvil-, cone-or drop-like cuticular projections were arranged in wedge-like fashion. The atrial chamber was almost spherical with a diameter of 138.4 m. Five main tracheal trunks of different luminal diameter as well as numerous channels opened into the atrial chamber.     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

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.     

Genetic transformation of the pathogenic fungus Wangiella dermatitidis

Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10–203 HmB-resistant colonies/g pAN7-1 on medium containing 100 g HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20–40 g/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17–50 transformants/g vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.     

A study of the vascular arrangement in the rat adrenal gland using non-radioactive microspheres

Summary The intra-glandular vascular arrangement in the adrenal has been studied using non-radioactive microspheres injected by three different routes: in-vivo injection into the left ventricle under pentobarbital anesthesia, postmortem orthograde, and postmortem retrograde injection. The doses of microspheres were 10⁵ (average size 24.7 m), 10⁶ (15.8 m) and 10⁷ (9.9 m). The entrapment rate of microspheres by the medulla as compared with the whole gland was measured in the serially sectioned tissue (section thickness 60 m).The entrapment rates of 25-m microspheres differed between the orthograde and retrograde injections, while the entrapment rates for 15-m microspheres were essentially similar irrespective of the route of injection.Our results support the conclusion from previous microangiographic studies that the adrenal cortex and medulla are supplied by different arteries but have a common venous outflow, and that direct communication between cortical and medullary sinusoids is not likely. The medullary blood flow per gram tissue weight is estimated to be larger than cortical blood flow.     

Assignment of the genes for human lysosomal acid lipases A and B to chromosomes 10 and 16

Summary This paper gives the results of studies on the effects of malathion on human lymphocytes stimulated by PHA, including cell survival, chromosomal aberration and nucleic acid content. Increasing malathion doses (10–70 g/ml) were introduced into cultures of human lymphocytes at different times relative to the time of PHA addition.At a concentration of 10 g/ml, malathion induced an increase in the RNA and DNA fractions in lymphocyte cultures. Higher malathion concentrations (50 and 70 g/ml), especially the dose of 70 g/ml, induced both a significant decrease in the content of RNA and DNA and reduced survival of cultured cells. The insecticide damaged the chromosome structure, but no correlation was revealed between the dose and the level of chromosome aberrations.     

Nicotonic and muscarinic components in acetylcholine stimulation of porcine adrenal medullary cells

Adrenal medullary chromaffin cells secrete catecholamines (CA) in response to cholinergic receptor activation by acetylcholine (ACh) released from splacnic nerve terminals. In cultured bovine chromaffin cells nicotinic receptors play a preponderant (> 90%) role in the control of CA release. By contrast, we found and report here that up to 40% of the ACh-evoked CA secretion from cultured porcine chromaffin cells can be associated with muscarinic receptor activation. The following results support our belief that in porcine adrenal medullary cells ACh (100 M) evoked CA secretion is mediated by both nicotinic and muscarinic cholinergic receptors. 1) Hexamethonium (100 M), a nicotinic receptor antagonist, inhibited ACh-induced CA secretion to ca. 40% of the control release and atropine (1 M), a muscarinic receptor antagonist, inhibited to ca. 60% of the control value. 2) We also found that ACh (100 M) evoked intracellular Ca2+ concentration ([Ca2+]i) rise was inhibited by these receptor antagonists to a different extent, and reversibly reduced by lowering the concentration of Ca2+ in the external medium ([Ca2+]o). This last maneuver ([Ca2+]o < 0.1 M) per se caused a marked reduction in the peak phase of the [Ca2+]i rise evoked by ACh (40% of the control response). Switching the external medium back to physiologic [Ca2+]o in the continued presence of ACh caused a partial recovery of the elevated [Ca2+]i. This [Ca2+]o-dependent [Ca2+]i rise was blocked by hexamethonium (100 M) but not by atropine (1 M). Conversely, the ACh-evoked [Ca2+]i rise in low external [Ca2+]o was blocked by atropine but not by hexamethonium. From these data we conclude that in porcine adrenal medullary cells an important fraction (ca. 0.4) of both ACh-induced CA secretion and peak [Ca2+]i rise is due to muscarinic receptor activation.     

Stimulatory effects of jasmonic acid on potato stem node and protoplast culture

The effect of jasmonic acid (JA), in a wide range of concentrations (0.01–100 M), on the development of potato plantlets (Solanum tuberosum L.cv. Vesna) was investigated in order to discriminate between physiological and supraoptimal effects of this growth regulator in vitro. Concentrations from 0.1–1 M significantly increased the length of developed plants. Application of JA in these concentrations resulted in a very well differentiated root system with many lateral branches. With increasing JA concentrations, the main roots shortened and thickened. Concentrations higher than 10 M led to the compaction of the stem, roots, and root hairs, giving a stunted appearance to the plants. The effect of JA on cell wall regeneration and callus formation was observed in potato leaf protoplast culture. JA at concentrations from 0.01–1 M stimulated cell division and microcalli development.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

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.     

Cellular organization of the embryonic lobster heart

Summary The cellular organization of the embryonic heart of the lobster Homarus americanus was examined in 6-week and 6-month-old animals. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Present in close association with the myocardium are cardiac neurons, hemocytes and so-called storage cells.Adjacent fibroblasts form fasciae adhaerentes and gap junctions. Adherent junctions also occur between fibroblasts and myocardial cells. Intercalated discs and differentiated membrane regions of close apposition (4 nm) occur between adjacent myocardial cells.The cardiac neurons form a ganglion that contains four small and five large somata. Regions of neuropil are present. Motor axons arising from the cardiac ganglion form neuromuscular synapses with the myocardial cells.The storage cells contain large inclusions and form gap junctions with the myocardial cells. They may supply nutritive material to the developing myocardium.The heart at 6 weeks is about 200 m long and 160 m wide. At 6 months, it is about 300 m long and 250 m wide. The myocardium at 6 weeks is one cell layer thick, and the cells are from 2–6 m in maximum width. At 6 months the myocardium is 2–4 cells thick, and the cells are from 6–12 m in width. Therefore, the myocardium grows by an increase in the number and size of the myocardial cells.     

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.     

Micropropagation of Hemidesmus indicus (L.) R. Br. through axillary bud culture

A procedure for rapid in vitro propagation of the aromatic and medicinal plant Hemidesmus indicus (L.) R.Br. (Family Asclepiadaceae) from nodal explants is described. The highest shoot multiplication rate of 8.2 ± 0.4 shoots/explant with a 95% frequency was achieved in S weeks culture period on Murashige and Skoog medium supplemented with 1.15 M kinetin and 0.054 M -naphthaleneacetic acid. Excised shoots were rooted on the same basal medium supplemented with 1.15 M kinetin and 7.35 M indole-3-butyric acid. Shoots derived from subcultures exhibited better rooting response than those from primary cultures. After a hardening phase of 2 weeks, there was a 70% transplantation success in the field.Abbreviations MS Murashige and Skoog (1962) medium - BA N⁶ benzyladenine; KN kinetin - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid