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.     

Diurnal patterns of skeleton formation in Pocillopora damicornis (Linnaeus)

Scanning, transmission and X-ray microanalytical electron microscopy were used to investigate the skeleton, organic matrix and calicoblastic ectoderm of the reef coral Pocillopora damicornis over a diurnal cycle. All skeletal surfaces, both during day and hight, are fasciculate except for skeletal spines on the branch tip apex at night where small (0.5 m) fusiform crystals are deposited. X-ray microanalysis shows that the fusiform crystals and needle-shaped crystals that compose the fasciculi are distinct forms of calcium carbonate. Demineralization of the skeleton reveals an organic matrix with two components which are related to the formation of fusiform crystals and fasciculi. During the day the calicoblastic ectoderm overlying all skeletal surfaces is 1–3 m thick. At night ultrastructural evidence suggests that skeletal deposition occurs only on those skeletal spines at the branch tip apex which are growing parallel with the branch growth axis. The calicoblastic ectoderm overlying apical skeletal spines at night shows a greater degree of cellular activity, and is thicker, than calicoblastic ectoderm overlying both other skeletal surfaces at night (<8 m cf. >6 m) and all skeletal surfaces during the day (<8 m cf. >3 m). The deposition of fusiform crystals on skeletal spines at the branch tip apex is proposed to promote deposition of fasciculi during the day, relative to other skeletal surfaces, providing a mechanism determining apical growth of branch tips. The results are discussed with respect to previous concepts of skeletal deposition in scleractinian corals.     

Adventitious shoot formation and plant regeneration from tissues of tomatillo (Physalis ixocarpa Brot.)

Cultured hypocotyl explants of tomatillo (Physalis ixocarpa Brot.), were evaluated with regard to their morphogenic responses to combinations of benzyladenine (BA, 0–5 M) with either naphthaleneacetic acid (NAA, 0–50 M) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0–50 M). The induction of shoots or roots was dependent on the cytokinin/auxin combination.Hypocotyl explants failed to form shoots when they were grown on media containing either a cytokinin or an auxin alone. The highest frequency of shoot formation was observed on media containing 12.5–25 M BA and 5 M NAA. Likewise the highest frequency of root formation was observed on media supplemented with 1 M BA and 1 M NAA. Complete plants were regenerated and transferred to soil, where they reached maturity.     

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     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish     

Replication of chromosomal DNA in cultured abnormal human cells

Summary The replication of chromosomal DNA in a series of abnormal human cell cultures has been studied by means of DNA-fiber autoradiography. In lymphocytes with trisomy 21, in fibroblasts of 45,X; 47,XXX; 49,XXXXY; and 49,XXXXX chromosomal constitution, and in fibroblasts from a patient with xeroderma pigmentosum (De Sanctis-Cacchione syndrome), the rate of DNA replication does not differ from that in normal cells, varying in a single fork from 0.2 to 1.0 m/min with a mean of about 0.6 m/min. In fibroblasts with trisomy 7 the rate of DNA replication is greater, varying from 0.3 to 1.2 m/min with a mean of about 0.8 m/min. The sizes of replication units in all cells examined are from 80 to 500 m with a mean of about 200–300 m.     

A combined micro-and semi-micro colorimetric determination of long-chain fatty acids from plant cutin

Summary Re-examination of the colorimetric fatty acid determination with copper nitrate, followed by complex formation with DIECA has shown that the method is not reliable if applied as described by Duncombe (1962, 1963): The Cu concentration is too high, the DIECA concentration much too low and the wavelength chosen (440 m) is suitable only for very low fatty acid concentrations.According to the results reported here the following alterations have to be adopted: The concentration of the copper nitrate solution should be 3%, a 0.5% solution of DIECA in butanol has to be used and measurements should be done at 492 m. The method described here offers the opportunity to determine fatty acid concentrations in the semi-micro range by measuring the filtered chloroform phase directly at 691 m, covering a range between 175 g/ml to 1.2 mg/ml. If the concentration turns out to be lower than 200 g F. A./ml, the same sample can be used for a micro-determination (up to 200 g/ml) at 492 m, after formation of the yellowish-brown complex by addition of 0.1 ml 0.5% butanolic DIECA solution to 1.0 ml of the chloroform phase.The method has been applied to determine the amount of free F. A. in cutin layers and cutin powder, revealing that the latter contains 5.6 times more free F. A. than the intact material. The free F. A. within the polymer seem to serve as interconnections for the main units of the cutin polylipid.     

Method of isolation of cysteine constitutive mutants of the cysteine regulon in Salmonella typhimurium.

Summary Size and shape of mitochondrial DNA molecules of Schizosaccharomyces pombe were analyzed by electron microscopy. Besides numerous linear molecules, circular molecules ranging from 0.83 m to 12.81 m were found. Depending on the method of preparation, both closed and open circular molecules were found. Most of the circular molecules could be assigned to five major size classes of 0.83±0.05 m, 1.7±0.05 m, 4.74±0.04 m, 5.74±0.04 m, and 8.32±0.07 m. Possible explanations for the different size classes of mitochondrial DNA molecules are discussed.     

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.     

Nicotinic receptor-elicited sodium flux in rat pheochromocytoma PC12 cells: Effects of agonists,antagonists, and noncompetitive blockers

Nicotinic agonists stimulate²²Na flux in rat pheochromocytoma PC12 cells. The stimulatory effect of carbamylcholine is maximal at 1 mM, while the stimulatory effect of nicotine and anatoxin maximize at the same level at 100 M and 10 M, respectively. The tertiary amines arecolone and isoarecolone have no effect on flux at 100 M, while the methiodides at 100 M stimulate flux to an extent similar to 1 mM carbamylcholine. Dihydro and alcohol analogues of isoarecolone methiodide have markedly smaller effects on flux. A preincubation for 2 to 20 min with carbamylcholine (2 mM), nicotine (300 M), anatoxin (30 M) or isoarecolone methiodide (100 M) causes marked desensitization to a subsequent carbamylcholine-elicited stimulation of flux. d-Tubocurarine, mecamylamine, hexamethonium, and chlorisondamine inhibit carbamylcholine-elicited flux with IC₅₀ values of 1.0, 0.8, 43, and 0.020 M, respectively. Atropine has no effect at 1 M, but reduces the response to carbamylcholine by 50% at 8.6 M, presumably as a noncompetitive blocker. Other noncompetitive blockers of nicotinic acetylcholine-receptors, such as histrionicotoxins, gephyrotoxin, pumiliotoxin C, phencyclidine, bupivacaine and piperocaine, inhibit carbamylcholine-elicited stimulation of²²Na flux with IC₅₀ values from 0.3 to 1.8 M. In contrast to d-tubocurarine, which inhibits carbamylcholine-elicited desensitization, and mecamylamine, which has no apparent effect on desensitization, chlorisondamine and certain noncompetitive blockers appear to enhance desensitization. The effects of agonists, antagonists and noncompetitive blockers at the neuronal nicotinic acetylcholine receptor-channel of PC12 cells are compared to their effects on binding of [¹²⁵I]-bungarotoxin to agonist-recognition sites and of [³H]perhydrohistrionicotoxin to noncompetitive blocker sites of the nicotinic acetylcholine receptor-channel of electric ray (Torpedo) electroplax membranes. There are marked differences in relative potencies for the two types of nicotinic acetylcholine receptor-channel.     

Pre-epithelial mucus layer in the colon of conventional and germ-free rats

Summary The pre-epithelial mucus layer (PML) and epithelial mucins were studied by mucin histochemistry in 10m-thick celloidinstabilized cryostat sections in the proximal and distal colon of conventional and germ-free rats aged 120 and 350 days. No continuous PML was found in the proximal colon. A continuous mucus blanket, of fairly homogenous thickness, was observed in the distal colon, where the PML-thickness was 40±24 m at 120 days of age and 44±22 at 350 days of age in conventional rats, and 25±17m (120 days) and 22±10 (350 days) in germ-free rats. The stainability of the PML by periodic acid-Schiff and Alcian Blue at pH 2.5 and 1.0 was stronger in conventional rats than in germ-free rats, indicating higher concentrations of mucosubstances and of acid and sulphated mucins, respectively. The PML of the conventional rat distal colon showed a stratified structure of up to eight sublayers. In the distal colon of germ-free rats, the whole gut wall thickness was reduced 47% compared to the conventional rat (germ-free: 185±73m, conventional: 350±115m). No stratification of the PML was observed. The presence of intestinal microflora obviously had a strong influence on the thickness, compactness, mucin content, mucin composition and structure of the pre-epithelial mucus layer.     

The units of DNA replication in the mammalian chromosomes: Evidence for a large size of replication units

The replication of chromosomal DNA in human and Chinese hamster cell populations has been studied by means of the DNA fiber autoradiography. It was found that the rate of DNA replication for one fork in human cells varies from 0.2 to 0.9 m/min, the average being 0.6 m/min. In the Chinese hamster cells the rate of DNA replication is greater, varying from 0.3 to 1.2 m/min, the average being 0.8 m/min. There are no clusters containing a great number of replication units in human and Chinese hamster cells. Sequences consisting of two or three replicons which belong to single DNA molecule have been observed, but their frequency was relatively low. The distances between the initiation points in such sequences of replicons vary from 40 to 280 m, the average value being 130 m. This value represents the minimum size of the replication units which have completed the DNA synthesis within 3 h of the S-period. The DNA synthesis in most replication units fails to be accomplished within the three hours of labelling. The process can be completed only in the fragments of DNA molecules of 40 to 200 m (the average value being 100 m) in human cells, whereas in the Chinese hamster cells the fragments of 40 to 250 m (the average being about 140 m) are completely replicated. Provided that the replication is bidirectional the complete replicons are supposed to contain two such fragments. Consequently, the greater part of replication units in mammalian cells covers the pieces of a few hundred microns in DNA molecules. The relation between replication process at the DNA molecules level and that at the metaphase chromosome level is discussed.     

Determination of copper, manganese and zinc in human liver

Autopsied liver tissue samples collected from 42 males and 31 females were analyzed for copper, manganese and zinc using atomic absorption spectrometry (AAS). With the exception of two liver samples for which the copper levels were determined to be 74.8 and 104.0 g/g (dry weight), hepatic copper concentrations were found to range from 1.7 to 32.4 g/g with a mean concentration of 14.2 g/g and standard deviation of 7.0 g/g. Manganese concentrations (with the exception of one sample having 12.9 g/g) ranged from 0.22 to 4.6 g/g with a mean of 2.26 ± 1.00 g/g. Hepatic zinc levels averaged 118.3 ± 44.4 g/g and ranged from 38.5 to 231.3 g/g. There were no apparent trends for the levels of any metals versus age nor were there any differences in average hepatic metal concentrations for males and females. © Rapid Science 1998.     

Karyometry of nuclei in liver cells

Summary In untreated adult male albino rats nuclear volume and the percentage of binucleate cells were determined in the first layer of hepatocytes adjacent to hepatic venous branches of varying diameters (<40 m, 40 m–80 m, 80 m–120m, 120 m–160 m, >160 m), and in the third and fourth layer of hepatocytes in the remainder of the perivenous parenchyma. In the first layer of hepatocytes adjacent to the vascular structures means of nuclear volume are significantly lower and percentage of binucleate cells significantly higher than in the cells of the remainder of the perivenous parenchyma. Within each area measured distribution curves of nuclear volume classes were homogeneous but showed heterogeneity in comparison with each other. The morphometric data presented in this study strongly support the opinion of the heterogeneity of liver cells in the perivenous zone, as previously postulated on the basis of histochemical investigations.Supported by the Deutsche Forschungsgemeinschaft (Hi 318/2-1)     

Locus-control-region-coupled betaS Antilles- and alpha2-hemoglobin genes select for high alpha2-hemoglobin expression in adult transgenic mice

Two transgenic lines of mice were produced which contained the ^S _Antilles- and ₂-hemoglobin genes trandemly coupled to the micro locus control region (LCR). The LCR^S _Antilles2-hemoglobin transgenic mice expressed high levels of ₂-hemoglobin while ^S _Antilles-hemoglobin expression was virtually undetectable. Abundant ₂-hemoglobin protein was observed in the blood of transgenic mice, while ^S _Antilles-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of -thalassemic mice. The LCR^S _Antilles2 transgenic mice demonstrate that if the LCR is coupled to the ^S _Antilles- and ₂-hemoglobin genes in tandem, only the distal ₂-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching.     

Coccidia of Brazilian edentates: Eimeria cyclopei n.sp. from the silky anteater,Cyclopes didactylus (Linn.) and Eimeria choloepi n.sp. from the two-toed sloth,Choloepus didactylus (Linn.)

Summary Eimeria cyclopei n.sp. is described from the silky anteater, Cyclopes didactylus, from Pará State, north Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in seven days at 26 to 28°C. Oocysts are ellipsoidal to sub-spherical, with a mean size of 28.1 × 23.6 m: the wall is 1.5 to 2.0 m thick, apparently with an outer thin, colourless membrane and two inner, thicker, striated and yellowish layers. There is no micropyle, oocyst residuum or polar body. The mean measurements of sporocysts are 19.0 × 9.0 m, and they are slightly asymmetrical, elongate pear-shape, with a plug-shaped Steida body projecting beyond the end of the sporocyst. Sporozoites are as long as or longer than the sporocysts: The sporocyst residuum is scattered between sporozoites in younger specimens and becomes condensed into rounded mass in older ones. The endogenous stages occur in the epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Uninucleate meront, microgamont and macrogamont precursors are recognizable morphologically. Mature meronts are 20.0 × 15.7 m some produce 12 to 20 merozoites which are 8.7 × 2.0 m, and others 10 to 26 merozoites which are 11.4 × 2.0 to 15.0 × 3.0 m. Mature microgamonts which are 27.5 × 24.1 m, produce from 150 to 170 microgametes of 7.1 × 1.0 m: microgametes have two flagella of unequal length. Mature macrogamonts are 28.4 × 24.5 m Eimeria choloepi n.sp. is recorded from the two-toed sloth, Choloepus didactylus, from the same area of Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in 23 days at 26 to 28°C. Oocysts with a mean size of 23.0 × 20.3 m, have a wall 2.0 to 2.5 m thick which is composed of two thick, yellowish and striated outer layers and a delicate, colourless inner one. There is no micropyle, oocyst residuum or polar granule. Mature sporocysts with a mean size of 11.3 × 7.1 m, are ellipsoidal to egg-shaped and have a poorly developed Steida body. The sporocyst residuum is composed of a small number of large globules: The sporozoites are longer than the sporocyst and strongly recurved. The endogenous stages occur in epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Dimorphic meronts produce 8 to 18 merozoites which are either 13.0 × 2.0 m or 13.0 × 3.0 m. Microgamonts produce 50 to 80 microgametes of 8.0 × 1.0 m. Mature macrogamonts are 18.3 × 17.9 m. ac]19820212     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

High frequency transformation of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid by electroporation

Summary A strain of Alcaligenes eutrophus producing poly--hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×10²/g DNA when 50 l competent cells at 10¹⁰/ml were pulsed by 11.5 kV/cm for 5 ms with 1 g DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.     

Procedures for the axenic isolation of conchocelis and monospores from the red seaweed Porphyra yezoensis

In order to maintain axenic seedstock cultures axenically of thecommercially important red seaweed, Porphyra yezoensis, aprocedure was developed for axenic isolation and culture of conchocelis andmonospores. For axenic isolation of the conchocelis, contaminated microalgaewere most effectively removed by filtering contaminated samples through a100-m mesh after sonication. Removal of bacteria and otheralgaewas accomplished using a mixture of 5 agents (0.02% chitosan, 100 gml^–1 GeO₂, 10 gml^–1 ampicillin, 40 gml^–1 kanamycin and 200 gml^–1 streptomycin). Axenic single colonies wereisolatedfrom a semi-solid medium prepared from 1% transfer gel. After collectingmonospores from the 40–50% density layer on a percoll-gradient, removalofbacteria and fungi from the monospores was accomplished using a mixture of 5antibiotics (3.5 g ml^–1 nystatin, 2 mgml^–1 ampicillin, 400 gml^–1 kanamycin, 50 gml^–1 neomycin and 800 gml^–1 streptomycin). Axenic single juvenile blades wereisolated from a semi-solid medium prepared from 0.5% transfer gel.     

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.