Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Anaerobic degradation of trans-cinnamate and ω-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a β-oxidation mechanism

The mechanism responsible for the initial steps in the anaerobic degradation of trans-cinnamate and -phenylalkane carboxylates by the purple non-sulphur photosynthetic bacterium Rhodopseudomonas palustris was investigated. Phenylacetate did not support growth and there was a marked CO₂ dependence for growth on acids with greater side-chain lengths. Here, CO₂ was presumably acting as a redox sink for the disposal of excess reducing equivalents. Growth on benzoate did not require the addition of exogenous CO₂. Aromatic acids with an odd number of side-chain carbon atoms (3-phenylpropionate, 5-phenylvalerate, 7-phenylheptanoate) gave greater apparent molar growth yields than those with an even number of side-chain carbon atoms (4-phenylbutyrate, 6-phenylhexanoate, 8-phenyloctanoate). HPLC analysis revealed that phenylacetate accumulated and persisted in the culture medium during growth on these latter compounds. Cinnamate and benzoate transiently accumulated in the culture medium during growth on 3-phenylpropionate, and benzoate alone accumulated transiently during the course of trans-cinnamate degradation. The transient accumulation of 4-phenyl-2-butenoic acid occurred during growth on 4-phenylbutyrate, and phenylacetate accumulated to a 1:1 molar stoichiometry with the initial 4-phenylbutyrate concentration. It is proposed that the initial steps in the anaerobic degradation of trans-cinnamate and the group of acids from 3-phenylpropionate to 8-phenyloctanoate involves -oxidation of the side-chain.Abbreviation 3-PP 3-phenylpropionic acid - 4-PB 4-phenylbutyric acid - 5-PV 5-phenylvaleric acid - 6-PH 6-phenylhexanoic acid - 7-PH 7-phenylheptanoic acid - 8-PO 8-phenyloctanoic acid - 4-P2B 4-phenyl-2-butenoic acid - GC/MS Gas chromatography/Mass spectrometry - HPLC High-pressure liquid chromatography     

Atomic force microscopy examination of the topography of a hydrated bacterial biofilm on a copper surface

A bacterium, designated CCI#8, that was isolated from a corroded copper coupon colonized both polished and unpolished copper surfaces under batch culture conditions. Atomic Force Microscopy (AFM) images revealed that the biofilm was heterogeneous in nature, both in depth and in cell distribution. Bacterial cells were shown to be associated with pits on the surface of the unpolished copper coupons. These observations support previous studies that CCI#8 is associated with the pitting corrosion of copper.     

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants

We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present a table for the conversion of old gene names to the new nomenclature.     

Appointments and awards

Elliot Meyerowitz the Biology Department, Caltech, and a member of the Board of Directors of the ISPMB, has been named Fellow of the American Academy of Arts and Science.     

Characterization of the central region containing the X-inactivation center and terminal region of the mouse X Chromosome using irradiation and fusion gene transfer hybrids

The irradiation and fusion gene transfer (IFGT) procedure provides a means of isolating subchromosomal fragments for use in the mapping of loci and for cloning probes from a particular area of a chromosome. Using this procedure, two large panels of somatic cell hybrids that contain mouse X Chromosome (Chr) fragments have been generated. These hybrid panels were generated by irradiating the monochromosomal mouse-hamster hybrid HYBX, which retains the mouse X Chr, with either 10 K or 50 K rads of X-irradiation followed by fusion with a recipient Chinese hamster cell line. IFGT hybrids retaining mouse material were generated at high frequency. These hybrids were used to orient loci in the X-inactivation center region that had not been resolvable in our interspecies backcross panel and also to map, within the terminal region of the X Chr, repeat elements detected by the probe p15-4. These hybrids not only complement existing interspecies meiotic mapping panels for the detailed analysis of specific regions of particular chromosomes, but also provide a potential source of material for chromosome-specific probe isolation.     

Differential effects of endothelin peptides in the systemic and pulmonary vascular beds of the cat

The cardiovascular and pulmonary responses to vasoactive intestinal contractor (VIC) were compared with those of endothelin (ET)-1, ET-2, ET-3 and sarafotoxin 6b (S6b) and the mechanism by which ET-1 alters vascular resistance was investigated in the hindquarters vascular bed of the cat. In a manner similar to ET-1 and ET-2, VIC at a dose of 0.3 nmol/kg i.v. produced increases in pulmonary arterial pressure (PAP) and biphasic changes in systemic arterial pressure (AP), systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR). The biphasic changes were characterized by initial decreases followed by increases. In contrast, ET-3 and S6b at doses of 0.3 nmol/kg i.v. produced mainly decreases in AP and SVR, increases in PAP, and biphasic changes in PVR. A monocyclic ET-1 analog and the ET-1 C-terminal hexapeptide fragment produced no effect on AP, SVR, PAP and PVR at doses of 30–100 nmol/kg i.v. ET-1 at a dose of 0.3 nmol i.a. produced a biphasic change in hindquarters perfusion pressure. The initial vasodilation and secondary vasoconstriction were not modified by a variety of blocking agents, whereas the vasoconstrictor response was significantly reduced by infusion of nimodipine, a calcium entry blocking agent. Results of the present study indicate that VIC, a peptide specific to the mouse gastrointestinal tract, elicits cardiovascular responses that are similar to those elicited by ET-1 and ET-2. The present results indicate that responses to these novel peptides are complex and while the mechanism of action remains uncertain, these data indicate that structural differences among the peptides confer differences in biological activity.     

Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students