Over-production of the D1:2 protein makes Synechococcus cells more tolerant to photoinhibition of photosystem II

Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp. PCC 7942 was studied using a tac promoter and the lacI ^Q system. Over-expression was induced with 40 g/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 mol photons m^-2 s^-1). This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein. The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres. When the cells were photoinhibited either at 500 or 1000 mol photons m^-2 s^-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG. These cells were also less prone to photoinhibition of PSII. It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance. This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment.     

Production of recombinant endostatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED₅₀) for recombinant endostatin was approximately 0.35 g ml^–1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l^–1 at 6 days of cultivation.     

Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.     

Establishment,Characterization, and Virus Susceptibility of a New Marine Cell Line from Red Spotted Grouper (<Emphasis Type="Italic">Epinephelus akaara</Emphasis>)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25°C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with . A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10^8.5 TCID₅₀ ml⁻¹), while the viral titer of frog Rana grylio virus 9807 (RGV₉₈₀₇) reached 10^3.5 TCID₅₀ ml⁻¹. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.     

Insect cells respiratory activity in bioreactor

Specific respiration rate ( ) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure . Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 × 10⁶ cells/mL and maximum specific respiration rate () of 7.3 × 10⁻¹⁷ molO₂/cell s. Drosophila melanogaster (S2) cells achieved 51.2 × 10⁶ cells/mL and of 3.1 × 10^–18 molO₂/cell s. S2AcGPV (expressing with rabies virus glycoprotein) reached 24.9 × 10⁶ cells/mL and of 1.7 × 10^–17 molO₂/cell s, while S2MtEGFP (expressing green fluorescent protein) achieved 15.5 × 10⁶ cells/mL and  = 1.9 × 10⁻¹⁷ molO₂/cell s. Relating to the Sf9, S2 cells reached higher maximum cell concentrations and lower specific respiration rate, which can be explained by its smaller size. These results presented useful information for scale-up and process control of insect cells.     

ATP-linked citrate lyase activity in the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum

Cell-free extracts of the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum strains 1C and L have been shown to cleave citrate with the formation of oxaloacetate and acetyl-CoA. This capacity was found in autotrophically grown cells as well as in the cells grown on media with acetate or L-glutamate. Citrate lyase activity in cell-free extracts is only measurable in the presence of citrate, adenosine-5-triphosphate, coenzyme A and Mg²⁺ or Mn²⁺. It is concluded on the basis of the obtained data that C. limicola f. thiosulfatophilum contains adenosine-5-triphosphate-linked citrate lyase (E.C.4.1.3.8). In contrast to green bacteria in the purple bacteria Ectothiorhodospira shaposhnikovii, Rhodospirillum rubrum and Thiocapsa roseopersicina citrate lyase activity was not found.     

Feeding rate inhibition in crowded Daphnia pulex

Feeding rates of Daphnia pulex fed a range of levels of the alga Chlamydomonas reinhardi of 15 °C are strongly density-dependent. At lower densities, Daphnia (30 1^–1) fed at higher rates than crowded (270 1^–1) Daphnia which manifest a relatively depressed saturation feeding response. At 30 individuals/liter, Daphnia consumed 8.5 – 15.7 × 10⁴ cells d^–1h^–1 (on a volume basis, 12.1 – 22.2 × 10⁶ m³), at 270 L^–1 3.7 – 3.9 × 10⁴ (5.2 – 5.5 = 10⁶ m³ cells d^–1h^–1 when feeding on algae at 80 000 cells ml^–1 (11.3 × 10⁶ m³ ml^–1). The feeding rate data best fit an Ivlev feeding function. An autoallelopath might be causing the repression. Water preconditioned with crowded Daphnia completely repressed feeding in uncrowded Daphnia after six hours.     

Reconstitution of an episomal mouse aprt gene as a consequence of recombination.

Summary When a functional murine adenine phosphoribosyltransferase (aprt) gene linked to bovine papilloma virus (BPV) DNA is transfected into Aprt^– L cells, the cells are rendered Aprt^– and the aprt gene persists as an episome.Cotransfection with two BPV vectors, one containing the 5 half of the aprt gene and the other the 3 half of the gene, that share about 300 by of common sequence in intron 2, produces Aprt⁺ cells with functional aprt as an episome. Southern blot analysis of low molecular weight DNA derived from Hirt extracts revealed the regeneration of a diagnostic Smal fragment, consistent with establishment of an episome with functional aprt that was reconstituted as a consequence of recombination. To establish cells with an episomal target for recombination, BPV vectors containing a G418 resistance marker and either the 5 half or 3 half of aprt were transfected into Aprt^– L cells. Stably transfected cells, selected by their growth in G418, were in turn transfected with DNA containing the other half of the aprt gene. Following selection of Aprt⁺ cells, Southern blot and polymerase chain reaction (PCR) analysis of low molecular weight DNA confirmed the presence of a complete episomal aprt gene. The region of DNA shared by the episomal aprt fragment and the transfected aprt half was sequenced after PCR amplification of the reconstituted, episomal gene and was found to be wild type. The region of overlap that serves as the substrate for recombination lies entirely within an intron and can, therefore, tolerate nucleotide substitutions and deletions. The absence of such errors in the sequences examined is consistent with recombination events that are not error prone.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

The role of cAMP in flagellation of Salmonella typhimurium

Summary A mutational alteration either in adenylate cyclase (cya ^-) or in cyclic-35-AMP (cAMP) receptor protein (crp ^-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation, cfs, partially suppressing the cya ^- mutation, was identified among the revertants of cya ^-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.     

Expression of recombinant rabbit UDP-GlcNAc:α3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I catalytic domain in Sf9 insect cells

UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyses a key reaction in the conversion of oligomannose to complex and hybridN-glycans. The cytoplasmic tail and transmembrane segment of rabbit GnT I cDNA were replaced with an in-frame cleavable signal sequence and the hybrid construct was inserted into the genome ofAutographa californica nuclear polyhedrosis virus (AcMNPV) under the control of the polyhedrin promoter. Sf9 insect cells were infected with the recombinant baculovirus and the enzymatically active and soluble catalytic domain of GnT I was purified from the medium (1–5 mg 1^–1) in two steps to a specific activity of abut 2 µmol min^–1 mg^–1 protein. Recombinant GnT I has been used for the chemical-enzymatic synthesis of analogues of Man1-6[GlcNAc1-2Man1-3]Man-O-octyl.Abbreviations AcMNPV Autographa californica nuclear polyhedrosis virus - FCS fetal calf serum - 1 µmol min^–1 international enzyme unit - MAG myelin associated glycoprotein - MOI multiplicity of infection - pfu plaque forming units - SDS-PAGE sodium dodecyl sulfate/polyacrylamide gel electrophoresis - Sf9 cells Spodoptera frugiperda insect cells - GnT I UDP-GlcNAc:3-d-mannoside 1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)     

Biochemical characterization of theO-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells

Alterations inN- andO-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. TheO-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoclonal antibodies to probe Western blots. TheO-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gal1-3GalNAc1-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human crythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAc1-Ser/Thr). Recombinant glycophorin A had the sameO-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect itsO-glycan composition. Deletion of theN-linked glycosylation site at Asn₂₆, by introducing the Mi.I mutation (Thr₂₈ Met) by site-directed mutagenesis, did not markedly affect theO-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells. This demonstrates that the presence or absence of theN-glycan did not influenceO-linked glycosylation of the recombinant glycoprotein. Finally, theO-glycans on recombinant glycophorin A expressed in the Lec 2 and Lec 8 glycosylation deficient CHO cells were characterized. TheO-glycans on Lec 2 cell glycophorin A were predominantly Gal1-3GalNAc1-Ser/Thr (T antigen), while those on Lec 8 glycophorin A were exclusively GalNAc1-Ser/Thr (Tn antigen). These results will lead to a better understanding of the cell biology and immunology of this important human erythrocyte glycoprotein.     

Response of Rhizobium leguminosarum to nickel stress

Rhizobium leguminosarum strain P-5 biovar viciae was sensitive to Ni²⁺ (MIC, 75 M) and showed concentration-dependent Ni²⁺ uptake in a wide concentration range (50–500 M). Ni²⁺ uptake up to a certain threshold limit also increased thiol content (66 nmol mg^–1 protein), proline content (10.85 nmol mg^–1 protein) and urease specific activity (500 nmol min^–1 mg^–1 protein) maximum corresponding to 100 M Ni²⁺ as the external concentration or 151 nmol Ni²⁺ mg^–1 protein as the intracellular buildup. Proline synthesis was stimulated most even at much lower Ni²⁺ concentration (25 M). Higher intracellular Ni²⁺ load neither favoured thiol nor proline biosynthesis nor urease activity. Ni²⁺ requirement of urease was ascertained by using EDTA-grown cells and the addition of bicarbonate (NaHCO₃, 100 mM) to the crude extract. The induction of thiol or proline by Ni²⁺, therefore, reflects the possible strategies adopted by bacterial cells to overcome the environmental stress.     

Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses

The cell-wall sulphated polysaccharide of the red microalga Porphyridium sp. has impressive antiviral activity against Herpessimplex viruses types 1 and 2 (HSV 1, 2) and Varicella zoster virus(VZV). Treatment of cells with 1 g mL^-1 polysaccharideresulted in 50% inhibition of HSV-infection as measured by the plaqueassay. Inhibition of the production of new virus particles was also shownwhen pre-infected cell cultures were treated with the polysaccharide. Inaddition, there was indirect evidence for a strong interaction between thepolysaccharide and HSV and a weak interaction with the cell surface.Depending on the concentration, the polysaccharide completely inhibitedor slowed down the development of the cytopathic effect in HSV or VZVpreinfected cells, but did not show any cytotoxic effects on Vero cells evenwhen a concentration as high as 250 g mL^-1 was used. Itseems therefore that the polysaccharide is able to inhibit viral infection bypreventing adsorption of virus into the host cells and/or by inhibiting theproduction of new viral particles inside the host cells. Thus, this alga seems tobe a good candidate for the development of an antiviral drug.     

Determination of an ethylene biosynthesis pathway in the unicellular green alga,Haematococcus pluvialis. Relationship between growth and ethylene production

In the freshwater ChlorophyceaeHaematococcus pluvialis, precursors of ethylene biosynthesis cycle are the same as those of higher plants: L-methionine S-adenosylmethionine 1-aminocyclopropane-1-carboxylic acid ethylene. However, the enzymatic complex of the last step of ethylene synthesis-ACCoxidase-differs from that of higher plants. It is stimulated by Co²⁺ (at least 10^-5 M), Mn²⁺ (at least 10^-6 M) and Ag²⁺ (at least 10^-4 M), inhibited by Cu²⁺ (at least 10^-5 M) and not affected by Zn²⁺, Fe²⁺ or Mg²⁺. ACCoxidase is also inhibited by salicylhydroxamic acid and by dark. Ethylene production is more important in young, mobile, green cells in active growth phase than in old, encysted and red cells in stationary growth phase. No peaks in ethylene production or respiration were observed during batch culture, as opposed to the situation with climacteric fruits.     

Molecular basis for erythrocyte Le(a+b+) and salivary ABH partial-secretor phenotypes: expression of a FUT2 secretor allele with an A→T mutation at nucleotide 385 correlates with reducedα(1,2) fucosyltransferase activity

TheSe ^wA385T mutation of the FUT2 gene was found to correlate with both the erthrocyte Le(a+b+) and/or salivary ABH partial-secretor phenotypes of Polynesians. Constructs with FUT1 and FUT2 wild type genes, and the FUT2Se ^wA385T,se ^G428A andse ^C571T mutated alleles, were cloned into pcDNAI, and expressed in COS-7 cells. COS-7 cells transfected with theSe ^wA385T allele had weak, but detectable, (1,2)fucosyltransferase activity, with an acceptor substrate pattern similar to the wild type FUT2 gene. Comparative kinetic studies from cell extracts with mutatedSe ^wA385T and wild type FUT2 alleles gave similarK _m values, but less enzyme activity was present in cells transfected withSe ^wA385T (V _max 230 pmol h^–1 mg^–1), as compared to those transfected with FUT2 (V _max 1030 pmol h^–1 mg^–1), suggesting that the mutated enzyme is more unstable. These results confirm that the molecular basis for the erythrocyte Le(a+b+) and the associated ABH salivary partial-secretor phenotype, is an amino acid change of Ile 129Phe in the secretor (1,2)fucosyltransferase.Abbreviations (1,3/1,4)fucosyltransferase GDP-L-fucose:-D-N-acetylglucosaminide 3/4--L-fucosyltransferase - (1,2)fucosyltransferase GDP-L-fucose: -D-galactoside-2--L-fucosyltransferase - bp base pairs - FUT1 H gene; FUT2,Se gene - FUT3 Lewis gene or Fuc-TIll gene - FUT4 Fuc-TIV gene - FUT5 Fuc-TV gene - FUT6 Fuc-TVI gene - MAb monoclonal antibody - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - se ^G428A FUT2 nonsecretor GA mutation at nucleotide 428 - se ^C571T FUT2 nonsecretor CT mutation at nucleotide 571 - Se ^wA385T FUT2 secretor weak AT mutation at nucleotide 385 - SSP sequence specific primer     

Proton translocation during denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Proton translocation during the reduction of NO ₃ ^- , NO ₂ ^- , N₂O and O₂, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO ₂ ^- to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO ₃ ^- , N₂O or O₂ was added to cells in the dark or with these compounds and NO ₂ ^- in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. The proton extrusion stoichiometry ( ratios) in illuminated cells were as follows: NO ₃ ^- 0.5N₂, 4.82; NO ₂ ^- 0.5N₂, 5.43; N₂ON₂, 6.20; and O₂H₂O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO ₃ ^- , NO ₂ ^- or N₂O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption, ratios without a permeant ion were NO ₃ ^- NO ₂ ^- ,-1.95; NO ₂ ^- 0.5 N₂O,-3.03 and N₂ON₂,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH reduced benzyl viologen - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DIECA N, N-diethyl-dithiocarbamate - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NEM N-ethylmaleimide     

A Kdp-like,high-affinity,K+-translocating ATPase is expressed during growth of Rhodobacter sphaeroides in low potassium media

Cells of the purple non-sulphur bacterium Rhodobacter sphaeroides express a high-affinity K⁺ uptake system when grown in media with low K⁺ concentrations. Antibodies againts the catalytic KdpB protein or the whole KdpABC complex of Escherichia coli crossreact with a 70.0 kDa R. sphaeroides protein that was expressed only in cells grown in media with low K⁺ concentrations. In membranes derived from R. sphaeroides cells grown with low K⁺ concentrations (induced cells), a high ATPase activity could be detected when assayed in Tris-HCl pH 8.0 containing 1 mM MgSO₄. This ATPase activity increased upon addition of 1 mM KCl from 166 to 289 mol ATP hydrolysed x min^-1 x g protein^-1 (1.7-fold stimulation). The K⁺-stimulated ATPase activity was inhibited approximately 93% by 0.5 mM vanadate but hardly by N,N-dicyclohexylcarbo-diimide (DCCD). These results indicate that the inducible K⁺-ATPase in R. sphaeroides resembles the Kdp K⁺-translocating ATPase of Escherichia coli. This Kdp-like transport system is also expressed in R. capsulatus and Rhodospirillum rubrum during growth in media with low K⁺ concentrations suggesting a wide distribution of this transport system among phototrophic bacteria.Abbreviations electrical potential difference across the cytoplasmic membrane - pH pH difference across the cytoplasmic membrane - BSA bovine serum albumine - PAGE polyacrylamide gel electrophoresis - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - PMSF phenyl-methyl-sulfonyl fluoride - DCCD N,N-dicyclohexylcarbodiimide - AIB 2--aminoisobutyric acid - TMG methyl--d-thiogalactopyranoside     

Resistance ofHER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared toHER2/neu-nonexpressing lines.HER2/neu ⁺ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in cold target inhibition experiments.HER2/neu ⁺ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and ₁ integrins was comparable to that ofHER2/neu ^– targets. In contrast, theHER2/neu ⁺ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis ofHER2/neu ^– carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression onHER2/neu ⁺ cells contributes to their resistance. The modest binding and lysis ofHER2/neu ⁺ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 onHER2/neu ⁺ targets. The following also supported deficiencies in post-binding events whenHER2/neu ⁺ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin,HER2/neu ⁺ cell lines were still resistant to LAK cytolysis, and (b)HER2/neu ⁺ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance ofHER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.Supported by research funds of the Veteran's Administration, the California Institute for Cancer Research and Jonsson Cancer Center core grant CA 16042 funded by NIH     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid