Scanning electron microscopy of rat maturation ameloblasts

Summary Post-secretory, maturation-phase ameloblasts were studied by scanning electron microscopy of freeze-fractured or dry-dissected rat incisors. These cells are in contact with the enamel which they secreted at an earlier time and which undergoes a process of continuing mineralization. The lateral intercellular compartment between maturation ameloblasts is sometimes continuous with the intercellular space of the papillary layer of the enamel organ, but often closed by basal ring contacts which correspond to terminal bars seen in transmission electron microscopy. The distal poles of the cells sometimes possess striated borders. Lateral cell surfaces may show longitudinal gutter-like depressions between ridges from which numerous intercellular connections arise; or a maze of lateral folds and ridges; or they may have mostly microvillous surface projections bordering a minimal intercellular space compartment. Preliminary correlations of groupings of basal, lateral and distal cell features indicate that basal-closed plus distal striated border cells may show every type of lateral surface. Cells without a striated border, whether open or closed basally, have ridge or maze lateral surfaces bordering a wide intercellular compartment. Basal-open plus striated border cells have microvillous or maze-like surfaces. These combinations of features are encountered a few times along the length of the maturation zone of individual incisors and suggest the existence of cyclical changes in the type of activity of maturation ameloblasts.     

Origin of α-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster and their functional relationship in the α-glycerophosphate cycle

The basis for the differentiation of l-glycerol-3-phosphate dehydrogenase (-GPDH) into larval and adult isozymes in Drosophila melanogaster was investigated by the correlation of a lack of appearance of each isozyme during development within Drosophila bearing -GPDH null alleles and by the study of a putative conversion factor. Conversion studies indicate the presence of a heat-labile RNase-resistant conversion factor present in crude larval extracts with the ability to convert GPDH-1 to GPDH-2 and GPDH-3 but not vice versa. In addition, null mutations at the Gpdh locus obliterate all isozymatic species of -GPDH in all developmental stages. These observations suggest that all -GPDH isozymes are the product of a single structural gene and that the multiple forms of this enzyme arise during successive developmental stages through an epigenetic modification of the primary Gpdh ⁺ polypeptide. Finally, observations are reported which bear on the functional divergence of the -glycerophosphate cycle in the adult and larval stage of development.This investigation was supported in part by NIH Research Grant No. GM-15691 and Genetics Training Grant No. 2 TI GM-685 at the University of North Carolina and by NIH Research Grant No. GM-11546 at North Carolina State University.Paper No. 5054 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Organogenesis versus embryogenesis from long-term suspension cultures of cucumber (Cucumis sativus L.)

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 M 2,4-D + 5 M BA, and (II) alternately three cultures in medium containing 5 M NAA + 5 M BA and one culture in 5 M 2,4-D + 5 M BA. After plating on solid medium with 0.5 M KIN + 0.1 M IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 M KIN + 0.1 M IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.Abbreviations BA 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Uptake of norepinephrine by isolated neurons of rat pontine reticular formation

Summary Neurons isolated from the nucleus reticularis pontis caudalis and the lateral vestibular nucleus of the rat brain stem have been incubated in buffered sucrose containing norepinephrine. Norepinephrine accumulated intracellularly was then visualized by fluorescence microscopy after formaldehyde condensation.Incubation in sucrose containing 1.0 g/ml norepinephrine resulted in a 2-fold increase in fluorescence that was blocked by 1×10^–5M cocaine. Maximal enhancement of fluorescence, accomplished by incubation of isolated neurons with 40 g/ml norepinephrine, was unaffected by cocaine but effectively blocked by 1×10^–3M metanephrine. Accumulation of norepinephrine was unaffected by pretreatment of animals with reserpine and nialamide.These properties of norepinephrine uptake suggest a neuronal membrane transport system for norepinephrine in isolated neurons similar to Uptake 2 which occurs extraneuronally. Although the intracellular localization of exogenous norepinephrine accumulated is not known, some concentration of norepinephrine in the perikaryal cytoplasm is suggested.This work was supported by U.S. Public Health Service Research Grant NB 07044. The competent technical assistance of Mrs. Gisela Griffith and Miss Susan J. Decker is acknowledged with gratitude.     

Structure of root nodules formed by Rhizobium on the non-legume Trema cannabina var. scabra

The structure of the nodules formed by Rhizobium on the non-legume Trema cannabina var. scabra was studied using the light microscope. The overall features of the nodules showed greater resemblance to the non-legume rather than the legume nodule. Nodule squashes yielded bundles of infection threads and bacteroids with morphological differences from rhizobial cells grown on yeast-mannitol-glucose agar. Two types of cell infection occurred within the bacterial zone; plant cells were either, like legumes, filled with rhizobia released from the infection threads (less than one third of infected cells) or were filled with the extensive growth and development of the infection thread. The rate of nitrogen fixation in the Trema nodule was high. It seemed that host cells filled with threads were active in N fixation.     

Histochemical sub-types of three fibre-types of avian skeletal muscle

Summary The soleus muscle of adult chicken was studied histochemically. Succinic dehydrogenase (SDH) and myofibrillar ATPase reaction, with or without preincubation in K₂-EDTA salt, were compared in serial frozen sections. Based upon the distributions of the above reactions, the three major fibre types distinguished were Type I red, Type II red and Type II white. On the basis of non preincubated ATPase reaction alone two sub-types of type I red fibres could be distinguished. However, following preincubation in a Cold solution of K₂-EDTA, Type II red fibres fell into two sub-types and Type II white fibres fell into three sub-types. Amalgamating the two already existing classifications, a more elaborate classification is presented for characterizing these different sub-types. The presence of two different or a spectrum of staining variations in a seemingly homogeneous population of muscle fibres in a given fibre-type emphasizes the possible correlation between this histochemical data and the heterogeneity of contraction times of the different motor units.     

Variation of glucose 6-phosphate dehydrogenase in the HeLa and Detroit 98 cell lines and their clonal derivatives

Contrary to other reports, we have found that the A type G6PD found in two permanent cell lines—HeLa (Gey), with its single cell clonal derivative HeLa S3, and Detroit 98, with its four clonal derivative lines—is not a single variant but rather at least three different isozymes. One is heat stable with normal specific activity and normal A type electrophoretic migration, another is heat labile with normal specific activity and normal A type electrophoretic migration, and the third is heat labile with reduced specific activity and slightly slow A type electrophoretic migration. We also found that in a mosaic cell population with respect to G6PD phenotype, the predominant G6PD phenotype varied randomly over a 5-month period, that the G6PD phenotype might be mutable in permanent cell lines, and that spontaneous human cell lines might not be HeLa cell contaminants as has been suggested.Aided by the National Institutes of Health General Research Support Grant # 5 S01 FR05507.     

Long-term carcinogenesis studies on 2,3,7,8-tetrachlorodibenzo-p-dioxin and hexachlorodibenzo-p-dioxins

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,6,7,8- and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxins (HCDDs) are among the most toxic and carcinogenic of man-made chemicals. These dioxins, as well as many of the other polychlorinated dibenzodioxins (PCDDs) and dibenzofuran (PCDFs) derivatives, are chlorinated aromatic compounds which are chemically stable, insoluble in water, and highly soluble in fats and oils.TCDD acts as a complete carcinogen in several species, causing both common and uncommon tumors at multiple sites. It is a highly potent chemical carcinogen in chronic animal studies, producing carcinogenic effects in laboratory animals with doses as low as 0.001 µg/kg/day. In rats, TCDD induces neoplasms in the lung, oral/nasal cavities, thyroid and adrenal glands, and liver. In mice, TCDD induces neoplasms in the liver and subcutaneous tissue, thyroid gland, and thymic lymphomas. In hamsters, it induces squamous cell carcinomas of the facial skin. Tumors of the integumentary system are reported after oral (mice and rats), intraperitoneal (hamsters), and dermal (mice) administration. A mixture of HCDDS (defined as the mixture of the 1,2,3,6,7,8- and 1,2,3,7,8,9 isomers used in the NTP experiments) are potent liver carcinogens in mice and rats.Pharmacokinetic studies in laboratory animals indicate that 50–90% of dietary TCDD is absorbed. It concentrates in adipose tissue and the liver. In mammals, the TCDD present in the liver is slowly redistributed and stored in fatty tissue. Elimination of TCDD occurs via excretion of metabolites in the bile and urine and passively through the gut wall. Metabolism is slow: the biological half-life of TCDD varies from weeks (rodents) to years (humans), and is strongly dependent upon the rate of TCDD metabolism.Many of the toxic effects of TCDD, including teratogenicity, may arise by receptor-mediated mechanisms. The induction of cytochrome P-448 and related enzymes by TCDD occurs by such a mechanism, and is related to the binding of TCDD to the Ah receptor. The specific mechanism(s) by which TCDD exerts its carcinogenic effects is unclear: receptor-binding may be part of the story. The role of the Ah receptor has been indicated in a skin promotion assay. The evidence for mutagenicity is inconclusive. TCDD did not induce lethal mutations, chromosomal aberrations, micronuclei or sister chromotid exchanges in rodents treated in vivo, nor was it mutagenic to bacteria, but it did enhance transformation of mouse C3H 10T1/2 cells by N-methyl-N-nitro-N-nitrosoguanidine and was mutagenic to mouse lymphoma cells.Under the State of California's Safe Drinking Water and Toxic Enforcement Act of 1986 (Proposition 65), TCDD and HCDDs were listed as chemicals known to the state to cause cancer. The International Agency for Research on Cancer (IARC) has classified TCDD as a Group 2B carcinogen (possibly carcinogenic to humans). Similarly, the EPA classified TCDD and HCDDs as Group B2 carcinogens (probable human carcinogens); and the DHHS' National Toxicology Program placed TCDD into the category of substances which may reasonably be anticipated to be carcinogens for humans.     

Purification and properties of the β-glucosidase of a yeast capable of fermenting cellobiose to ethanol: Dekkera intermedia van der walt

Summary The intracellular enzyme -glucosidase of Dekkera intermedia was partially purified by ion-exchange chromatography and gel filtration. The molecular weight of this enzyme was 310,000; its optimum pH was 5.0 and optimum temperature was 55°C. This enzyme was active against different -glucosides and was inhibited by p-chloromercurybenzoate, gluconolactone, and glucose. A glucosyltransferase activity appeared in the presence of ethanol. The biosynthesis of this enzyme was constitutive but repressed by glucose.     

Physiology of polyol formation by free and immobilized cells of the osmotolerant yeast Pichia farinosa

Summary Some physiological data of cells of Pichia farinosa immobilized on sintered glass Raschig rings were compared with data from free cells. Glucose consumption and productivity of total polyols (arabitol, glycerol and erythritol) showed a simultaneous inter-lag phase. Enzymes that catalyse steps of the pentosephosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transaldolase and polyol dehydrogenase) showed a distinct increase after transfer of the cells into production medium. The activity of glycerol-3-phosphate dehydrogenase was generally low. Only alcohol dehydrogenase presented the inter-lag phase mentioned above.Offprint requests to: H.-J. Rehm     

On the subsarcolemmal localization of phenazine ethosulfate-linked α-glycerophosphate dehydrogenase activity in pigeon pectoralis white muscle fibres

Summary In this study frozen sections of avian striated muscles were incubated for mitochondrial -glycerophosphate dehydrogenase (-GPD) reaction, and the effect of menadione, phenazine methosulfate (PMS) or phenazine ethosulfate (PES) as intermediate electron acceptors was evaluated. Under histochemical conditions, PMS or PES-linked -GPD reaction was poor in the chicken posterior latissimus dorsi and chicken pectoralis muscles. However, PMS or PES-linked -GPD reaction was present characteristically in ths subsarcolemmal mitochondria of the broad white fibres of the pigeon pectoralis muscle only; the subsarcolemmal mitochondria of the narrow red fibres lacked such a reaction pattern. The above reaction pattern, however, differed when compared with the menadione-linked -GPD reaction. The present histochemical evidence suggests the existence of an inherent heterogeneity in the mitochondrial populations of the different avian striated muscle fibres studied.     

Carotenoids of Rhizobia

With increasing concentrations in the growth medium of the cyclization inhibitors nicotine or 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) the previously identified bicyclic carotenoids of Rhizobium lupini (2,3,2,3-tetrahydroxy-,-caroten-4-one and 2,3,2,3-tetrahydroxy-,-carotene) were successively replaced by hitherto unknown monocyclic carotenoids. By application of mass and nuclear magnetic resonance spectroscopy 3 carotenoids were identified as 2,3-trans-dihydroxy-,-caroten-4-one, 2,3-trans-dihydroxy-,-carotene, and 3-hydroxy-,-caroten-4-one. A further compound was tentatively established as (2- or 3-)monohydroxy-,-carotene. It was found that other inhibitors such as diphenylamine or 4-chloro-5-(dimethylamino)-2-,,(trifluoro-m-tolyl)-3-(2H)-pyridazinone (San 6706) did not affect the pigment pattern. The results are discussed in relation to carotenoid biosynthesis in Rhizobium lupini.Abbreviations CPTA 2-(4-chlorophenylthio)-triethylamine hydrochloride - San 6706 4-chloro-5-(dimethylamino)-2-,,-(trifluoro-m-tolyl)-3-(2H)-pyridazinone     

Purification and properties of two fructose-6-phosphate phosphoketolases in Bifidobacterium

Fructose-6-phosphate phosphoketolase was purified from type strains of two species of the genus Bifidobacterium: B. globosum and B. dentium. The first species has a preferred animal habitat, like feces of animals and rumen of cattle; the latter is harboured in human habitats, like feces and dental caries of man. Two electrophoretic types of phosphoketolase (F6PPK) were previously distinguished and called animal and human type according to the habitat of the bifid organism. The purified preparations of these two phosphoketolases displayed very different optimum pH range, metal activator and molecular weight; outstanding difference was found in the substrate specificity: the enzyme from B. globosum was able to split xylulose-5-P as well as fructose-6-P, whereas the phosphoketolase from B. dentium appeared to be specific for fructose-6-P.     

A successive histochemical staining for succinate dehydrogenase and “reversed”-ATPase in a single section for the skeletal muscle fibre typing

Summary A procedure is described which simplifies the classification of skeletal muscle fibres in that it allows a simultaneous evaluation of both the oxidative capacity and the intensity of reversed ATPase of the fibres, and thus enables to distinguish three fibre types — SO, FOG and FG — in one tissue section. After preincubation at pH 4.1–4.2 the cryostat section is incubated for succinate dehydrogenase (SDH) and subsequently for reversed-ATPase. This is followed by the fixation with neutral buffered formaldehyde. The results of typing of chicken, minipig and rabbit fibres in a single muscle section stained with this technique are identical to those obtained with the usual method based on a comparison of serial sections of which one is stained for SDH activity the other for reversed-ATPase activity.     

Polysaccharide and protein secretion by grass microhairs

Summary Secretory activities of bicellular microhairs from grasses belonging to the subfamilies Chloridoideae, Arundinoideae, Panicoideae, and Bambusoideae, and including the chloridoid, panicoid and Enneapogon microhair morphological types, have been investigated. Light microscopic histochemistry indicated that all microhairs studied secrete polysaccharide and protein (or glycoprotein), including those which also secrete salt. Localization of polysaccharide at ultrastructural level using periodic acid-thiocarbohydrazidesilver proteinate staining revealed that in panicoid type microhairs dictyosomes are involved in polysaccharide secretion, whereas in the chloridoid and Enneapogon types partitioning membranes seem to be involved instead.Abbreviations Ag silver precipitates representing localization of polysaccharide - BC basal cell - C cuticle - CC cap cell - CH cuticular chamber - CN system of membrane bound channels and vesicles - CP chloroplast - CW cell wall - D dictyosomes - M mitochondria - N nucleus - PTM partitioning membranes - RER rough endoplasmic reticulum - S secretory material - St starch grain - US unstained dictyosome cisternae - V vesicle     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

Isolation and characterization of plant myosin from pollen tubes of lily

Summary A plant myosin was isolated from pollen tubes of lily,Lilium longiflorum. Pollen tubes were homogenized in low ionic strength solution containing casein, and myosin from this crude extract was purified by co-precipitation with F-actin prepared from chicken breast muscle, followed by hydroxylapatite column and gel filtration column chromatography. Upon SDS-PAGE on 6% polyacrylamide gel, only 170 kDa polypeptide was detected in the purified myosin fraction. Furthermore, with immunoblotting using antiserum raised against 170 kDa polypeptide, only the 170 kDa component crossreacted in the crude sample of pollen tube proteins. This antiserum did not crossreact with the heavy chain of skeletal muscle myosin. The ATPase activity of pollen tube myosin was stimulated up to 60-fold by F-actin prepared from chicken breast muscle. The translocation velocity of rhodamine-phalloidin-labeled F-actin on a glass surface covered with pollen tube myosin ranged from 6.0 to 9.8 m/s with an average of 7.7 m/s. This velocity was similar to or a little faster than that of the cytoplasmic streaming that occurred in pollen tubes. These results suggested that myosin composed of a 170 kDa heavy chain produces the motive force for cytoplasmic streaming in pollen tube of lily.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazin-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate     

Gene flow in an almond orchard

Summary Gene flow by pollen between trees is essential for nut set in commercial almond orchards, due to the self-incompatibility of almond cultivars used. A study of gene flow has been carried out in an orchard composed of single rows of a pollinating cultivar between every double row of the most commercially desirable cultivar, Nonpareil. This two-to-one planting pattern was repeated throughout the orchard, and several pollinating cultivars were used in various parts of the orchard in an attempt to provide flowers for cross-pollination with Nonpareil at all stages of flowering of the latter. Using isozyme markers GPI-2, LAP-1, AAT-1, PGM-1, and PGM-2 and three newly-defined isozyme markers for almond — IDH, G6PD, and SDH — it has been shown that the gene flow resulting in nut set is quite restricted, taking place most strongly between neighboring halves of cross-compatibile pairs of trees. Even that half of a tree facing away from the pollinating tree has significantly less gene flow to it, while the next tree further on has few nuts set by fertilization from the pollinating tree under consideration. This result is surprising considering the comparatively large distances that the honeybee brought into the orchard in large numbers must travel within the orchard. To explain this apparent paradox and the observation that in most cases only a small proportion (<20%) of flowers set nuts, it is suggested that the honeybee predominantly visits only one cultivar, flying along the row of the cultivar to do so, and that cross-pollination results from accidental or rare visits involving two or more compatible cultivars.     

Anaerobic energy metabolism of goldfish,Carassius auratus (L.)

Summary The concentrations of pyruvate, lactate, oxalo-acetate, aceto-acetate -hydroxybutyrate, -ketoglutarate, glutamate, NH ₄ ⁺ , NAD⁺ and NADH were measured in goldfish tissues after previous conditioning to normal and anoxic (12h) conditions. For 11 different metabolites efficiency of different extraction methods was tested by means of internal standards. The recoveries were generally over 80%. The substrate/product couples of the reactions catalysed by lactate dehydrogenase, malate dehydrogenase, -hydroxybutyrate dehydrogenase and glutamate dehydrogenase were used as redox parameters. In the lateral red muscle the redox state did not change during 12 h of anoxia. In the dorsal white muscle only the cytoplasmic redox state underwent a change, as indicated by the increase of the lactate/pyruvate ratio from 20 to 110. In liver both cytoplasm and mitochondria were reduced during anoxia. From the measured values the NAD⁺/NADH ratio was found to change only in white muscle, while the calculated free NAD⁺/NADH ratios were reduced in anoxic white muscle cytoplasm, anoxic liver mitochondria, and anoxic liver cytoplasm. Oxalo-acetate concentrations calculated from the equilibrium constants of lactate dehydrogenase and malate dehydrogenase were at least one order of magnitude smaller than the measured values. The data obtained from anoxic goldfish are in contrast to available data on other animals and support earlier reports which indicate that this animal has a special anaerobic metabolism. The results are discussed especially with respect to the role of ethanol as a sink for reducing equivalents.Abbreviations LDH lactate dehydrogenase - MDH malate dehydrogenase - HBDH -hydroxybutyrate dehydrogenase - GIDH glutamate dehydrogenase