Microelectrode-recorded development of the symplasmic autonomy of the sieve element/companion cell complex in the stem phloem of Lupinus luteus L.

From the cambial stage onwards, the symplasmic autonomy of sieve element/companion cell complexes (SE/CC-complexes) was followed in stems of Lupinus luteus L. by microinjection techniques. The membrane potential and the symplasmic autonomy of the mature SE/CC-complex was measured in successive internodes. A microelectrode was inserted into SE/CC-complexes or phloem parenchyma cells (PPs) and, after stabilization of the membrane potential, the membrane-impermeant fluorescent dye Lucifer Yellow CH (LYCH) was injected intracellullary. The plasmodesmata of the cambial SE/ CC precursor were gradually shut off at all interfaces beginning at the walls to be transformed into sieve plates. In the course of maturation, symplasmic discontinuity was maintained at the longitudinal walls of the complex. In the transverse walls of the SE, wide sieve pores were formed giving rise to longitudinal multicellular symplasmic domains of SE/CC-complexes. Symplasmic isolation of the files of mature SE/CC-complexes was demonstrated in several ways: (i) the membrane potential of the SE/CC-complexes (between -100 mV and -130 mV) was consistently more negative than that of the PPs (between-50 and -100 mV), (ii) No exchange of LYCH was observed between SE/CC-complexes and the PPs. Lucifer Yellow CH injected into the SEs exclusively moved to the associated CCs and to other SE/CC-complexes whereas LYCH injected into the PPs was only displaced to other PPs. (iii) The electrical coupling ratio between adjacent PPs was ten times higher than that between SE/CC-complex and PP. A gradient in the membrane potential of the SE/CC-complexes along the stem was not conclusively demonstrated.Abbreviations LYCH Lucifer Yellow CH - membrane potential - PMF proton-motive force - PP phloem parenchyma cell - SE/CC-complex sieve element/companion cell complex - SR-G sulphorhodamine G     

Intraprotoplasmic feruloylation of arabinoxylans in Festuca arundinacea cell cultures

Graminaceous primary cell walls contain polysaccharides to which are esterified feruloyl residues. Ester biosynthesis is highly specific and the present experiments were performed to ascertain the likely site of feruloylation in living grass cell cultures. Cell cultures of tall fescue grass (Festuca arundinacea Schreber) incorporated exogenous l-[1-³H]arabinose into polymers at a linear rate after a short lag of approx. 1–3 min. Radiolabelled polymers did not start to accumulate in the culture medium until 20–35 min after [³H]arabinose was supplied. However, polymer-bound feruloyl-arabinose residues began to accumulate ³H after a lag of 1–3 min. Assuming that the onset of secretion of radiolabelled polymers into the medium indicates the time before which essentially all the radiolabel was internal to the plasma membrane, the results show that the polysaccharide-bound [³H]arabinose residues must have been feruloylated within the protoplast.Abbreviations AIR alcohol-insoluble residue - BAW butan1-ol/acetic acid/water (12:3:5 by volume) - BEW butan-1-ol/ ethanol/water (20:5:11 by volume) - EPW ethyl acetate/pyridine/ water (8:2:1 by volume) - R_Ara Chromatographic mobility relative to that of l-arabinose We are very grateful to Mr. Gundolf Wende for assistance with the characterisation of the feruloyl esters. K.E.M. is funded by a studentship from the Science and Engineering Research Council in collaboration with Zeneca Agrochemicals.     

Inhibition of Bacillus spores by combinations of heat,potassium sorbate,NaCl and pH

Viability of spores of Bacillus cereus was totally inhibited at 85°C over 30 min by adding 0.4% (w/v) potassium sorbate with 6% (w/v) NaCl at pH 4.5. Viability of B. stearothermophilus spores was totally inhibited at 95°C for 45 min in a buffer at pH 4.2 containing 0.8% (w/v) potassium sorbate and 8% (w/v) NaCl. A synergistic inhibitory effect was demonstrated in some of the combinations. The inhibition may be due to interference with the heat-resistance apparatus of the spores.O.B. Oloyede was and J. Scholefield is with the Department of Bioscience & Biotechnology, Food Science Division, University of Strathclyde, Glasgow G1 1SD, UK. O.B. Oloyede is now with the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria.     

Suppression of lymphocyte spontaneous proliferative response by proteolipid protein peptide in patients with HAM/TSP

To understand the immune mechanism suggested in HTLV-I-associated myelopathy (HAM/TSP), we investigated T cell response to proteolipid protein (PLP). Because of high autologous proliferative response (APR) of peripheral blood mononuclear cells (PBMC) in culture, the lymphocyte proliferation assay was not useful in this disease. Unexpectedly, however, APR was profoundly (70–98%) suppressed in 6 of 9 cases when PLP peptide 105-124 was added in the culture. PLP peptide 85-104 or 145-159 also suppressed APR in a few cases. Time course study showed that the peptide-mediated suppression became apparent after day 4 in culture. The results can be interpreted as that suppressor cells recognizing the PLP peptides were present in the PBMC of HAM/TSP patients and suppressed the APR as the consequence of antigen specific response. This may indicate that a T cell response to certain PLP determinants is involved in the pathomechanism of HAM/TSP at least in part. Molecular mimicry between PLP and HTLV-I mayaccount for the T cell sensitization to PLP in HAM/TSP.Special issue dedicated to Dr. Marjorie B. Lees.     

Mycobacterial cell wall: Structure and role in natural resistance to antibiotics

Abstract Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents. The low permeability of the mycobacterial cell wall, with its unusual structure, is now known to be a major factor in this resistance. Thus hydrophilic agents cross the cell wall slowly because the myobacterial porin is inefficient in allowing the permeation of solutes and exists in low concentration. Lipophilic agents are presumably slowed down by the lipid bilayer which is of unusually low fluidity and abnormal thickness. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance, which requires synergistic contribution from a second factor, such as the enzymatic inactivation of drugs.     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Fractionation of bovine spermatozoa for sex selection: A rapid immunomagnetic technique to remove spermatozoa that contain the H-Y antigen

A study was conducted to rapidly fractionate bovine spermatozoa on the basis of cell-surface H-Y antigen (i.e., Y chromosome-bearing spermatozoa). A novel, rapid immunomagnetic method was developed for removal of spermatozoa that bound to anti-H-Y IgG. Fluorescent labeling and flow cytometry were used to measure the efficiency with which spermatozoa binding to anti-H-Y were removed by the immunomagnetic technique. Washed bovine spermatozoa (n=7 bulls) were treated with a mouse monoclonal IgG antibody to H-Y antigen (MoAb 12/49). Fluorescent labeled goat antibody against mouse IgG was added to label those spermatozoa with cell-surface H-Y antigens. Supermagnetized polymer beads coated with an anti-antibody to the MoAb 12/49 were then added to the spermatozoa. After 20 min of incubation, spermatozoa were exposed for 2 min to a magnet, causing the magnetized particles to adhere to the sides of the tube. Nonmagnetized spermatozoa in the supernatent were aspirated and analyzed for fluorescent label by flow cytometry. Approximately 50% of spermatozoa not subjected to immunomagnetic separation were fluorescent labeled, and about one-half of the spermatozoa were observed microscopically to be bound to the magnetized polymer beads prior to magnetic separation (P<0.05). Following magnetic separation, only 1.2% (P<0.05) of the spermatozoa in the magnetic supernatent were fluorescent labeled. Assuming that only Y chromosome-bearing spermatozoa have cell-surface H-Y antigens, the present immunomagnetic fractionation removed almost all of the Y chromosome-bearing spermatozoa, leaving a population that was greater than 98% X chromosome-bearing spermatozoa.     

Altered free calcium transients in pig kidney cells (LLC-PK1) cultured with penicillin/streptomycin

Summary The effect of a conventional antibiotic (penicillin/streptomycin) mixture on the widely used kidney epithelial cell line, LLC-PK₁, was investigated by measuring growth and intracellular free calcium. Free calcium concentration was the same in cells cultured for 3 to 7 wk with (“plus”) and without (“minus”) antibiotics both at rest and when challenged with high (14 mM) external calcium. When exposed to vasopressin, minus cells exhibited significantly smaller calcium transients than plus cells. A similar difference existed for transients elicited by a calcium ionophore, 4-br-A23187. After longer periods of culture (>20 wk), minus cells grew slower than plus cells but on reaching confluence (minus cells took 1 day longer) the morphologies and viabilities were indistinguishable. The finding that culture with penicillin/streptomycin reversibly modified some properties of LLC-PK₁ cells, at least partly through altered calcium homeostasis, is of importance for workers using this cell model to study drug effects and raises the general possibility of similar effects on other cultured cells.     

Caprine blood groups. 1. The B system

Twelve of 24 monospecific caprine reagents produced by absorption of alloimmune antisera identified a complex blood group system of goats which was designated B, based on the results of a small comparison test with ovine reagents. The frequencies of the 12 B factors differed significantly among the Australian Angora, Texan Angora, Cashmere, and Dairy goat breeds. Three of the antigens detected by the reagents were shown to be related as linear subtypes, designated Ba₁, Ba₂, and Ba₃, and inherited as alleles. The segregations of B factors in 80 sire groups involving 1086 offspring demonstrated that groups of B factors (phenogroups) segregated as products of allelic genes. This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, New South Wales 2031, Australia.