Uptake and Retention of Selenite and Selenomethionine in cultured K-562 cells

The selenium uptake and retention have been studied in K-562 cells exposed to selenite or selenomethionine. In the uptake experiments the cells were exposed to two doses of selenite (5 or 50 M) or selenomethionine (10 or 50 M). In the retention study the cells were treated for 2 h with the above mentioned doses of the selenocompounds before being observed at different times. The selenium uptake in cells exposed to selenite 5 M began to saturate at 8 h, but increased again between 48 and 96 h. In cells exposed to selenite 50 M the selenium uptake never reached a maximum, however, at 48 and 96 h the cell viability decreased strongly. The two doses of selenite showed different retention patterns, with a relatively small cellular decrease of selenium after treatment with selenite 5 M compared to treatment with 50 M of selenite. The selenium uptake in cells exposed to selenomethionine 10 M or selenomethionine 50 M began to saturate at 24 h and 48 h, respectively. The retention patterns were similar for both selenomethionine doses with a continuous decrease of the selenium concentration during the whole observation period. The results indicated a more controlled uptake and retention pattern of selenomethionine compared to selenite.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.     

Effects of the growth regulator 4PU-30 on growth,K+ content,and alkaloid production in tobacco callus cultures

Growth, K⁺ content, and alkaloid production were compared in nonorganogenetic callus cultures ofNicotiana tabacum cv. Burley 21 grown at 25°C in the dark on two different media: a basal medium with 1 M -naphthaleneacetic acid and 1 M kinetin, and one with 1 M -naphthaleneacetic acid and 1 M 4PU-30 (N-(2-chloro-4-pyridyl)-N-phenylurea). These callus tissues behaved differently not only in growth and K⁺ content but also in alkaloid production. In comparison to cultures grown with kinetin, those grown with 4PU-30 showed a significantly higher fresh weight and dry weight and K⁺ content during the growth period studied. The data clearly indicate a positive correlation between K⁺ uptake rate stimulated by 4PU-30 and cell enlargement rate. However, the alkaloid biosynthesis in the callus tissues was activated by the supply of kinetin and diminished by that of 4PU-30. It thus appears that cellular enlargement of meristematic tissue stimulated by 4PU-30 limited alkaloid production.     

Modulation of specific [3H]phenytoin binding by benzodiazepines

We have shown that diazepam (ED₅₀ 2.4 M), flunitrazepam (ED₅₀ 10.2 M) and Ro5-4864 (ED₅₀ 5 M) are able to enhance both total and specific [³H]phenytoin binding. Picrotoxin (IC₅₀ 1.43 M) and chloride, either NaCl or KCl (IC₅₀ 42.4 M) inhibit both the increase in total and specific binding of [³H]phenytoin, Ro 15-1788 does not. The optimum time for this enhancement was 3–4 hours. While the ED₅₀'s for the benzodiazepines are high their order of potency suggests that an involvement of both the peripheral type benzodiazepine receptor and the GABA-chloride ionophore complex is likely. Clonazepam (IC₅₀ 23 M), oxazepam (IC₅₀ 12 M) chlordiazepoxide (IC₅₀ 35 M) and Ro8682-10, a convulsant benzodiazepine (IC₅₀ 16 M) all inhibit both total and specific [³H]phenytoin binding. These effects were not blocked by chloride ions, picrotoxin or Ro 15-1788, and reached equilibrium within 45 minutes. This order of potency also parallels that for the peripheral benzodiazepine receptor in rat brain. These data suggest the presence of a micromolar benzodiazepine receptor site which may play a role in the control of CNS excitability. Nitrazepam, medazepam, bromazepam and the tetralobenzodiazepines U38335, U42794, U43434, and U37834 had no effect on total or specific [³H]phenytoin binding nor on the actions of the other benzodiazepines described in concentrations up to 50 M.     

The Effect of Phenylalanine on DOPA Synthesis in PC12 Cells

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The K_m for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the K_m for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.     

Assessment of membrane potential changes using the carbocyanine dye,diS-C3-(5): synchronous excitation spectroscopy studies

The fluorescence of the voltage sensitive dye, diS-C₃-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C₃-(5) fluorescence from cells and from the supernatant. It has been found that diS-C₃-(5) fluorescence in the supernatant can be selectively monitored at _exc = 630 nm and _em= 650 nm, while the cell associated fluorescence can be observed at _exc= 690 nm and _em = 710 nm. A modified theory for the diSC₃-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, _p=_p-°_p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K⁺ gradients. It has been demonstrated that the membrane potential change, _p,can be measured on an absolute scale. Offprint requests to: J. Plasek     

Toward a gibbsian approach to the problems of growth and cancer

Summary Certain sections ofJosiah Willard Gibbs's thermodynamics papers might be applicable to biological equilibrium and growth, normal or abnormal.Gibbs added terms _i dm _i to the differential of the internal energy d=td–pd, (t=temperature,p=pressure,=entropy,=volume) where is the potential of substancem _i , to provide for chemical as well as thermal and mechanical equilibrium. In this article a further generalization is suggested, to include biological equilibrium by adding to de terms of the form GdN, the variableN being the number of cells, where is a growth potential that measures exactly the resistance toward spontaneous growth. The functionG, like _i is intensive in nature (i.e. depends on intensive variables only) except for a conversion factor ,M=m _i , affording possible insight into why incipient abnormal growth is often independent of the number of cells. Useful applications might follow from identities between , or and or respectively. The following new function is studied, , a natural generalization of theGibbs free energy function , the possibility of measuring it electrically, and comparison of its role with that of for the possible experimental determination ofG. Gibbs's necessary and sufficient conditions for heterogeneous equilibrium ofn components inm phases are generalized and also modified to include broader restraining conditions like ,j=1,f,n, the > being characteristic of only living cellular phases. Careful appraisal of the term biological stability is followed by new criteria for stability, instability, and limits of stability, (neutral equilibrium) in terms of derivatives ofG, with possible medical applications. Three different sections of Gibbs's works tend to indicate that, for a biological phase, lower pressure usually increases its stability. The equation , where =surface tension,p, p = pressures,r, r=radii of curvature, is applied to possible control of tissue growth at interfaces. Methods of altering the equilibrum between three phasesA, B, C by varying the interfacial tensions _AB , _BC , _AC , using relations like _AB < _AC + _BC for stability of theA, B interface, suggest different means for shifting biological equilibrium between normal and abnormal cells through the introduction of new third phases at the interface. Various devices are mentioned for possible control of growth through proper channeling of surface or other equivalent forms of energy.     

Identification of phosphate granules occurring in seedling tissue of two palm species (Phoenix dactylifera and Washingtonia filifera)

Haustoria of two palm species, Phoenix dactylifera L. (date) and Washingtonia filifera (Lindl.) Wendl were carefully dissected from seeds, and the ultrastructural characteristics of the large, electron-opaque granules present in the cells of this tissue were compared using standard aldehyde and OsO₄ fixations. In Washingtonia, the granules were smaller than those in date and were more variable in size and presence of non-opaque inclusions. All granules appeared to be membrane bounded although they often filled the bounded space. No protein, lipid, carbohydrate or tannins were found in the granules by standard staining procedures. The granules stained positively with two different metallic-phosphate stains which contained either bismuth or lead. Energy dispersive X-ray microprobe analysis, done on aldehyde-fixed granules and those stained with both phosphate stains, confirmed the fact that phosphorus and calcium were present in the granules. The granules also bound the metallic stains as expected. All procedures consistently confirmed the presence of phosphate in the granules. The data are most consistent with the hypothesis that the granules are composed of polyphosphate.Abbreviations and symbols EDAX energy-dispersive X-ray microanalysis - K K shell peak - K K shell peak - L L shell peak - L L shell peak - M M shell peak     

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.Abbreviations EAT cells Ehrlich ascites tumour cells - na-EAT cells non-adherent EAT cells - a-EAT cells adherent EAT cells - c/m EAT cells cultured a-EAT cells passaged in mice - HPTLC high-performance thin-layer chromatography - PBS 10 mM phosphate-buffered saline, pH 7.2, containing 0.15 M NaCl - EDTA ethylene-diaminetetraacetic acid - TFA trifluoroacetic acid - TG thioglycollate - Cer ceramide (N-fatty acyl sphingosine) - GM3 NeuAc2-3Gal1-4Glc-Cer - GM2 GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GM1a Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GD3 NeuAc2-8NeuAc2-3Gal1-4Glc-Cer - GD1a NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GT1b NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Glc-Cer - LacCer Gal1-4Glc-Cer - Gb3 Gal1-4Gal1-4Glc-Cer - Gb4 GalNAc1-4Gal1-4Gal1-4Glc-Cer This paper is dedicated to my esteemed colleague, Sen-itiroh Hakomori on the occasion of his 65th birthday.     

The gibberellin synthesis inhibitors,ancymidol and flurprimidol,promote in vitro rooting of white pine microshoots

Summary Ancymidol and flurprimidol were tested for their ability to induce in vitro rooting on axillary proliferated shoots of white pine (Pinus strobus L.). Shoots were treated for 30 days (pulse) with growth regulators, then subcultured to 0.5X medium for conifer morphogenesis without growth regulators. A pulse treatment containing 5 M ancymidol and 0.54 M naphthaleneacetic acid resulted in 43% rooted shoots, whereas a pulse treatment with 0.54 M naphthaleneacetic acid alone resulted in 7% root formation. Flurprimidol also stimulated rooting of white pine shoots, but was less effective than ancymidol. No detrimental effects on shoot growth were observed with the gibberellin synthesis inhibitors at the 5 M concentration used. Some rooted shoots were successfully acclimatized to the greenhouse.Abbreviations ancymidol -cyclopropyl-(4-methoxyphenyl)-5-pyrimidinemethanol - flurprimidol -(1-methylethyl--[4-trifluromethoxy)phenyl]-5-pyridinemethanol - GA gibberellin - MCM medium for conifer morphogenesis - NAA 1-naphthaleneacetic acid     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Photoreversion und Photoprotektion bei UV-induzierten Mutationen des nichtphotoreaktivierbaren Bacteriums E.coli phr?

Summary Stationary phase cells of strain phr^–/MC 2 ofE. coli are not photoreactivable but the frequency of UV-induced mutations to low Streptomycine-resistance (S 3, 3/ml) is decreased strongly by illumination with light of fluorescence tubes (310 to 500 nm) after UV-irradiation. Also dark-reversion (DRM) of these mutations due to keeping UV-irradiated cells in saline is observed. Illumination before UV-irradiation decreases the frequency of the mutations (photoprotection against mutation=PPM) to the same extent as the combined action of photoreversion (PRM) and DRM. The lag-phase of cell division is prolonged strongly by illumination from 80 min without light to 150 min by the light-dose of highest activity. The additional lag is nearly the same if the illumination is done before, after or without UV-irradiation; this lag is about additive to the small lag caused by UV. Pre-illumination of the stationary-phase cells does not cause photoprotection against killing (PP), it even decreases the survival after high UV-doses. The observations support the hypothesis that PRM in this strain may be indirect, i.e. caused by the light-induced additional division lag which enhances the dark repair of UV-premutations. Also spontaneous premutations which are apparently present in the stationary-phase cells seem to be influenced by the light in this way.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

The chromosomal localization of human β-galactosidase revisited: a locus for β-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22

Summary A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme -galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental -galactosidase, it was observed that the structural locus for the -galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human -galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against -galactosidase, followed by analysis via gel electrophoresis and fluorography.The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the protective protein was previously shown to be intimately associated with human -galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of -galactosidase by aggregating -galactosidase monomers into high molecular weight multimes. Both chromosome 3 and 22 are therefore necessary to obtain normal levels og -galactosidase activity in human cells.     

Polarities in Mammalian Evolution Seen Through Homologs of the Inner Cell Mass

Embryogenetic pathways differ markedly among monotremes, marsupials, and placentals, and their analysis provides information of fundamental importance to recognition of mammalian evolutionary directions. The cap of cuboidal cells of the marsupial late unilaminar blastocyst, generally known as the embryonic area, probably is induced to form (prior to origin of Hensen's node) by signals from earliest hypoblastic cells (anterior visceral endoderm). The thickened cap is a medullary plate of sauropsid terminology because it includes epiblastic cells presumptive to neurectoderm (including neural crest), Hensen's node, primitive streak, and gut endoderm. The remainder of the definitive embryo (i.e., parts of epidermal origin, including ectodermal placodes) derives from squamous ectoderm (surrounding the medullary plate) of the blastocyst's ill-named trophoblastic area. Amniotic ectoderm develops farther distally within the trophoblastic area. The autapomorphic inner cell mass (ICM) of placental mammals is homologous to medullary plate of the marsupial blastocyst plus morphologically undefined, proximal parts of surrounding ectoderm (of the trophoblastic area). Considerations of early cell lineages in marsupials are greatly affected by recognition that the boundary between future embryonic and extra-embryonic tissues does not match the margin of the medullary plate (i.e., embryonic area). Marsupials and monotremes largely conform to sauropsid early embryogenesis, but placentals express, at earliest developmental stages, innovations unique within Amniota that are linked to early establishment of the brain. Neonatal marsupials and hatchling monotremes are extremely altricial and closely comparable anatomically/physiologically; they share a temporal pattern in combining early morphogenesis of craniofacial features (related to suckling) with deferral of telencephalic completion into postnatal/posthatching life. Placentals contrast greatly in establishing the central nervous system prior to rudiments of the cranial skeleton and associated musculature, and they complete essentials of forebrain development before birth. Comparative evidence from transitory periderm suggests that primordial eutherians had extremely altricial hatchlings or newborns, whichever was the mode of early development. Details remain unknown about the origin of the unique specialization of ICM plus encapsulating trophoblast from the more generalized blastula of ancestral synapsids.     

Allospecific T cell recognition of HLA-A2 antigens: Evidence for group-specific and subgroup-specific epitopes

Interleukin 2-dependent alloreactive cytotoxic T cell lines, with activity predominantly directed against the HLA-A2 antigen, have been generated in vitro by stimulating blood mononuclear cells from donors nonimmune to the Epstein-Barr (EB) virus with appropriate numbers of EB virus-transformed B cells from A2-homozygous individuals. Such effector cells were tested against a panel of EB virus-transformed target cell lines all expressing the serologically defined A2 antigen but typed into common A2 and variant A2 subgroups on the basis of their recognition by A2-restricted EB virus-specific cytotoxic T cells. Variant A2 responder cells cocultivated with common A2-bearing stimulators gave rise to effector T cell lines which recognized only the common A2-bearing subgroup of targets. By contrast, responder cells from A2-negative donors stimulated with common A2-bearing cells produced effector T cell lines in which the strong lysis of common A2-bearing targets was accompanied by a lower, but still significant, lysis directed against all targets within the variant A2 subgroup. In both cases, lysis of the target cells was blocked equally well by the anti-A2-specific monoclonal antibody MA2.1 as by the monoclonal antibody W6/32 specific for HLA-A, -B, and -C determinants. This suggests that HLA-A2 molecules possess at least two distinct sets of epitopes capable of inducing alloreactive T cell cytotoxicity: first, epitopes probably associated with T cell-restricting sites, which generate subgroup-specific responses, and second, epitopes shared by all A2 molecules, and perhaps associated with serologically defined sites, which generate pan A2 group-specific responses.Abbreviations used in this paper EB Epstein-Barr - IL-2 Interleukin 2 - UM unfractionated mononuclear - AET aminoethylisothiouroniumbromide hydrobromide     

Synthesis of selectively radiolabeled hexasaccharides for the determination of enzymatic regioselectivity

Poly-N-acetyllactosamines provide backbone structures for functional modifications such as sialyl Lewis X. To understand how the biosynthesis of poly-N-acetyllactosamines is regulated, two branched oligosaccharides of the structure Gal1,4GlcNAc1, 6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl 1 and 2 were synthesized in which one of the terminal galactose units was selectively radiolabeled. Hexasaccharides 1 and 2 were assembled from the chemically synthesized pentasaccharide precursors GlcNAc1,6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl3 and Gal1,4GlcNAc1,6(GlcNAc1, 2) - Man1,6 Man-octyl 4 respectively, through treatment with UDP-1-[³H]-Gal and 1,4 galactosyltransferase. Compounds 1 and 2 were subsequently incubated with UDP-GlcNAc and the UDP-GlcNAc: Gal1-4Glc(NAc)1,3-N-acetylglucosaminyltransferase (i-GlcNAc transferase) resulting in a partial conversion to a mixture of heptasaccharides which were purified by HPLC. The branch selectivity of the addition of N-acetylglucosamine to compounds 1 and 2 was then characterized by endo--galactosidase digestion of the heptasaccharides, followed by isolation of the resultant pentasaccharides on C18 reverse-phase silica cartridges. Comparison of the amount of radiolabel to a control reaction lacking endo--galactosidase indicated the favored site of GlcNAc addition to be the lower 1,2-branch over the 1,6-branch by a 3:1 ratio.     

Characterization of Aeromonas salmonicida variants with altered cell surfaces and their use in studying surface protein assembly

Aeromonas salmonicida variants were characterized for alterations in their cell surface structure and used to examine reconstitution of the surface protein layer (A-layer). Variants lacking outer membrane O-polysaccharide were devoid of A-layer and excreted stainable floret-like material of the surface protein (A-protein). One variant, showing partial loss of O-polysaccharide, was associated with a disrupted A-layer and excretion of some A-protein. Variants lacking A-protein but possessing O-polysaccharide rapidly absorbed and concentrated sufficient excreted A-protein at the cell surface to coat the cells with a single confluent layer. Although differences in electrophoretic mobilities of A-proteins and O-polysaccharides from typical and atypical strains were evident, the different A-proteins and A-protein-deficient variants were interchangeable for reconstitution of a surface protein layer. No association of A-protein with cell surfaces of unrelated gram-negative bacteria was observed.Abbreviations A-layer additional surface protein layer - A-protein surface protein - Ast Aeromonas salmonicida typical - Asa Aeromonas salmonicida atypical - A^- phenotypically A-protein-negative variant - O^- phenotypically O-polysaccharide-negative variant - O^wk phenotypically O-polysaccharide weak variant - BHI brain heart infusion - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TEM transmission electron microscopy     

In vitro plant regeneration from leaf-derived callus in ginger (Zingiber officinale Rosc.)

Excised tissues from young leaves of ginger cv. Maran were cultured on revised Murashige and Skoog medium supplemented with various concentrations of growth regulators. The presence of 2, 4-D in the culture medium at 9.0–22.6 M resulted in callus growth. Organogenesis and plantlet formation occurred when the concentration of 2,4-D is reduced to 0.9 M and with the addition of 44.4 M BA into the medium. The rate of plant regeneration increased when the growth regulators are completely removed from the culture medium in the subsequent subcultures. The plantlets developed extensive root systems when they were put in MS liquid medium with 5.4 M of NAA. The establishment of these plantlets in soil is about 80%.Abbreviations BA N⁶-benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid