Cation-exchange Fast Protein Liquid Research Articles

Protein, thermal and functional properties of α-, γ- and ω-gliadins of wheat and their effect on bread making characteristics

The study investigated the rheological, microstructural and functional properties of α-, γ- and ω-gliadins. Crude gliadin was subjected to cation exchange fast protein liquid chromatography and eluted individual gliadins in 17 peaks. NanoLC Electrospray ionization coupled MS-MS analysis revealed the identity of polypeptides from selected peaks as α-, α/β-, and γ-gliadins with high PLGS (ProteinLynx Global SERVER™) score. Inclusion of 1% (w/w) total gliadin containing, α-, γ- and ω-gliadins to base flour decreased dough development time and stability, while increased peak resistance and dough softening. The addition of gliadins improved the loaf volume of bread. DSC revealed multiple peak denaturation temperatures (Tp) for gluten, α- and γ-gliadins whereas, single peak for gliadins and ω-gliadins. The enthalpies of denaturation (ΔH) for gliadin and its purified α-, γ-, and ω-gliadin fraction were higher than gluten. Scanning electron micrographs (SEM) displayed collateral relationship among dough microstructure and mixing characteristics of these elements. These findings provided insight into the functionality of purified gliadins and potential use in the development of different food products.

Physico-chemical characterization of a milk-clotting fraction extracted from turkey (Meleagris gallopavo) proventriculus

Abstract Valorization of avian by-products such as turkey (Meleagris gallopavo) proventriculus in the preparation of milk clotting enzymes constitutes an alternative to commercial rennet. The clotting enzyme of Meleagris gallopavo has been prepared by ammonium sulfate at 50% saturation followed by purification by cation-exchange fast protein liquid chromatography (FPLC), and determination of the clotting protease activity performed on milk incubated at 35 °C. The clotting fractions are concentrated and dialyzed against Tris/HCl pH 7.0 through a membrane with cutoff 8000 Da, and separated by anion-exchange FPLC. The coagulating fractions obtained by cation-exchange chromatography are also separated by reversed-phase high-performance liquid chromatography, which revealed only one fraction able to coagulate milk. Electrophoresis analysis revealed a band with apparent molecular mass of 36.5 kDa and seemed to correspond to pepsin-like enzyme according to primary sequence alignment analyses. Maximal clotting activity was obtained for optimal conditions of temperature of 55 °C and pH 5.4.

Production of a Class II Two-Component Lantibiotic of Streptococcus pneumoniae Using the Class I Nisin Synthetic Machinery and Leader Sequence

Recent studies showed that the nisin modification machinery can successfully dehydrate serines and threonines and introduce lanthionine rings in small peptides that are fused to the nisin leader sequence. This opens up exciting possibilities to produce and engineer larger antimicrobial peptides in vivo. Here we demonstrate the exploitation of the class I nisin production machinery to generate, modify, and secrete biologically active, previously not-yet-isolated and -characterized class II two-component lantibiotics that have no sequence homology to nisin. The nisin synthesis machinery, composed of the modification enzymes NisB and NisC and the transporter NisT, was used to modify and secrete a putative two-component lantibiotic of Streptococcus pneumoniae. This was achieved by genetically fusing the propeptide-encoding sequences of the spr1765 (pneA1) and spr1766 (pneA2) genes to the nisin leader-encoding sequence. The chimeric prepeptides were secreted out of Lactococcus lactis, purified by cation exchange fast protein liquid chromatography, and further characterized. Mass spectrometry analyses demonstrated the presence and partial localization of multiple dehydrated serines and/or threonines and (methyl)lanthionines in both peptides. Moreover, after cleavage of the leader peptide from the prepeptides, both modified propeptides displayed antimicrobial activity against Micrococcus flavus. These results demonstrate that the nisin synthetase machinery can be successfully used to modify and produce otherwise difficult to obtain antimicrobially active lantibiotics.

Characterisation of whey proteins of camel ( Camelus dromedarius) milk and colostrum

Variation in the composition of whey proteins from camel ( Camelus dromedarius) colostrum and milk was recorded over a 192 h period following parturition. Whey proteins were separated by cation-exchange fast protein liquid chromatography and identified by polyacrylamide gel electrophoresis. The main components of whey proteins in camel milk and colostrum were similar to that in bovine, except for the lack in β-lactoglobulin. Serum albumin was the major whey protein present in camel milk, with an average concentration of 10.8 g/l. Camel colostrum was rich in immunoglobulins G, which consist of IgG1, and the enzyme inhibitory antibodies IgG2 and IgG3. The concentration of these proteins decreased rapidly 48 h post partum. Lactophorin (proteose peptone-component 3) and basic whey protein were detected only within 48 h after parturition, reaching a level of 4.9 and 3.1 g/l at 192 h post partum, respectively. The maximum level of lactoferrin (2.3 g/l) was observed at 48 h after parturition. Camel milk and colostrum were shown to be rich in protective proteins, especially IgG2 and IgG3, which revealed to be a potential source of inhibitory antibodies.

Endowing human pancreatic ribonuclease with toxicity for cancer cells.

Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.

Isolation ofMycobacterium Tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti-ES-31 antibody by affinity chromatography.

Proteins secreted into the culture medium byMycobacterium tuberculosis (M. tb) are shown to be source of antigens of immunodiagnostic interest. Anin vitro released 31 kDa antigen ESAS-7F isolated fromM.tb H(37)Ra culture filtrate by salt precipitation, SDS-PAGE and cation exchange fast protein liquid chromatography (FPLC) was shown earlier to be a diagnostically important antigen fraction. In this report, we describe the isolation of ESAS-7F antigen using monospecific antibody coupled to sepharose CL-4B column. The percentage recovery of ESAS-7F antigen using affinity chromatography was approximately 8% of the total ES antigen proteins compared to 0.05% obtained by conventional purification steps using salt precipitation, SDS-PAGE and FPLC. Similar seroreactivity was observed by the antigen isolated by both the methods in indirect ELISA. Affinity chromatography helped in an increased recovery of ESAS-7F antigen and obviates the need for time consuming conventional purification steps.

Cation-Exchange Purification of Mutagenized Bovine β-Casein Expressed in Transgenic Mouse Milk: Its Putative Asn68-Linked Glycan Is Heterogeneous

Bovine β-casein (A2 genetic variant) was mutagenized to (L70S/P71S) and expressed in transgenic mouse milk. This protein now carries the signal (N68S69S70S71) that mimics a consensus eukaryotic glycosylation signal (N-X-S/T) (3). Hypothetically this protein should be glycosylated at N68 by any eukaryotic organism producing it. This novel protein was purified from transgenic mouse milk by Mono-S cation-exchange fast protein liquid chromatography (FPLC). The novel β-casein was separated without cross contamination from mouse caseins by using acetate buffer (pH 5.0) in the presence of 6 M urea, octyl-glucopyranoside and 2-β-mercaptoethanol. The purified (L70S/P71S) β-casein showed an N-linked oligosaccharide attached to Asn68 and different lectin binding profiles compared with the same protein expressed in yeast. The mouse-expressed β-casein (L70S/P71S) was specific to Concanavalin A, wheat germ agglutinin, Erythrina cristagalli agglutinin, and Ulex europaeus, indicating its oligosaccharide structure is different in the mammary gland of mouse than the reported glycosylated β-casein expressed in Pichia pastoris (4). In addition, the five serine residues located at amino terminus of wild type bovine β-casein were shown to be normally phosphorylated as in native bovine β-casein.

Influence of some physico-chemical characteristics of commercial rennet caseins on the performance of the casein in Mozzarella cheese analogue manufacture

Abstract The compositional and chromatographic characteristics of ten rennet caseins and their relationships to rennet casein performance in Mozzarella cheese analogue manufacture were determined. Some compositional parameters (moisture, ash and calcium) were weakly related to performance, while cation-exchange fast protein liquid chromatography did not indicate differences between the caseins which could be related to performance in Mozzarella cheese analogue manufacture. When the rennet caseins were hydrated at 8.0% (w/w) protein for 120 min in 0–1.0% (w/w) disodium orthophosphate (DSP), the level of protein and calcium solubilised varied depending on the DSP concentration and on the particular rennet casein. Differences were also observed in the rates of protein and calcium solubilisation when each rennet casein was hydrated at 4.0% (w/w) protein in 0.5% (w/w) DSP for up to 120 min. Levels of protein and calcium solubilised and rates of solubilisation were in some instances found to relate to rennet casein performance in Mozzarella cheese analogue manufacture. These results suggest that calcium mediated protein–protein interactions in the different caseins are disrupted at different rates and to different extents during hydration at similar concentrations of calcium sequestering salt. These differences may arise due to protein and/or mineral (ash, calcium) modifications during heat treatment of skim milk prior to rennet casein manufacture or on heat treatment during manufacture; the extent of modification appears to subsequently influence the hydration and solubilisation of a rennet casein in DSP and its performance in Mozzarella cheese analogue manufacture.

Speciation of aluminium in forest soil extracts by size exclusion chromatography with UV and ICP-AES detection and cation exchange fast protein liquid chromatography with ETAAS detection

Aluminium speciation was studied in forest soil extracts by size exclusion chromatography (SE) with UV and inductively coupled plasma-atomic emission spectrometric (ICP-AES) detection and cation exchange fast protein liquid chromatography (FPLC) with ETAAS detection. Size exclusion chromatography was performed on a Superdex HR75 10/30 column. Isocratic elution with 0.15 mol dm −3 NaCl in TRIS–HCl buffer (pH=5.5) was applied over 100 min at a flow rate of 0.35 cm 3 min −1. The chromatographic run was followed at 278 nm and separated Al species also determined ‘off line’ in 0.875 cm 3 fractions by ICP-AES. The analytical procedure enabled speciation of high molecular weight Al complexes. Cation exchange FPLC was performed on a Mono S HR 5/5 column. Aqueous 8 mol dm −3 NH 4NO 3 linear gradient elution was applied over 10 min at a flow rate of 1 cm 3 min −1. Separated Al species were collected in 0.5 cm 3 fractions and Al determined ‘off line’ by ETAAS. The analytical procedure enabled speciation of some positively charged monomeric Al species. Negatively charged species were eluted with the solvent front. The combination of the two analytical techniques was successfully employed in speciation of Al in forest soil extracts. Water was used as an extracting solution. It was found experimentally that 80–95% of Al in aqueous extracts of forest soils exists in monomeric Al forms. Water soluble Al (30–40%) is bound to high molecular weight complexes with humic substances. The remaining monomeric Al in the low molecular weight fraction exists as AlF 2+, Al-oxalate and Al-citrate species.

Analysis of Major Caprine Milk Proteins by Reverse-Phase High-Performance Liquid Chromatography and Electrospray Ionization-Mass Spectrometry

Major proteins from caprine milk were separated by preparative gel permeation and cation-exchange fast protein liquid chromatography and were characterized by flow injection analysis by electrospray ionization mass spectrometry. In addition, proteins from whole skim milk and whole casein were analyzed by coupling reverse-phase HPLC and electrospray ionization mass spectrometry by two different chromatographic methods. These methods successfully resolved the major caprine milk proteins and main casein variants. The experimental molecular masses of major milk proteins and variants were: 19,302 for κ-CN 2P; 25,599 for αs2-CN A-11P; 25,514 for αs2-CN B-10P; 23,370 for αs1-CN A-8P; 23,345 for αs1-CN B-8P; 23,264 for αs1-CN E-8P; 18,817 for αs1-CN F-3P; 23,835 for β-CN 6P; 18,181 for β-LG; 14,180 for α-LA and 66,318 for serum albumin.

Isolation and characterization of a 31 kDa mycobacterial antigen from tuberculous sera and its identification with in vitro released culture filtrate antigen of mtb H37Ra bacilli.

Antigens released in vivo are of considerable interest in the immunodiagnosis of infectious diseases. Circulating antigen was isolated from bacteriologically confirmed tuberculous sera by ammonium sulphate precipitation. The protein fraction between 36%, and 75%, ammonium sulphate was reactive with tuberculosis (TB) sera showing the presence of circulating tubercular antigen (CTA). Fractionation of CTA on ultrogel AcA 34 gel filtration column gave 3 protein fractions CTA1, CTA2 and CTA3. CTA2 showed maximum antigenic activity by sandwich enzyme-linked immunosorbent assay (ELISA). SDS-PAGE fractionation and seroreactivity studies showed the presence of highly reactive tubercular antigen in CTA2-7 protein fraction by sandwich ELISA. Further fractionation of CTA2-7 on cation exchange fast-protein liquid chromatography (FPLC) gave 4 antigenic fractions, of which CTA2-7D was seroreactive similar to 31 kDa antigen (ESAS-7F) isolated from in vitro culture medium. Furthermore, CTA2-7D could inhibit binding of in vitro released ESAS-7F to affinity purified antibodies in inhibition ELISA. CTA2-7D antigen may be used as a target antigen in confirming active tubercular infection. Biochemical characterization showed circulating antigen CTA2-7D to be a lipoglycoprotein is released in vivo. ESAS-7F as a glycoprotein is released in vitro culture.

Determination of Anti-?-Gliadin Antibodies in Serologic Tests for Coeliac Disease

Serologic detection of coeliac disease in the general population or in subjects belonging to risk groups implies the use of a test with high efficiency, large-scale use, and low cost. The enzyme-linked immunosorbent assay (ELISA) technique is the most appropriate assay for performing this kind of studies. Even though anti-gliadin determination has been the test most frequently used as the first step in screening procedures, many false-positive results produced a low-specificity test. In a previous work a selective recognition of omega-gliadins, mainly by IgA antibodies, was observed. Results also indicated that omega-gliadins can be useful as antigens in serologic detection of coeliac disease. We therefore wanted to analyse the anti-gliadin antibody reactivity by using purified gliadins and to evaluate the actual performance of the anti-omega-gliadin antibody test. A population consisting of 105 coeliac patients, 81 healthy controls, and 73 subjects in a disease control group was analysed. Anti-endomysium (EMA), both IgG and IgA anti-omega-gliadins, and anti-tissue transglutaminase (tTG) antibodies were determined. Concordant results, positive and negative, in the EMA and IgG and IgA anti-gliadin determinations were observed in 220 of 259 samples from the total population analysed. The three assays showed high efficiency, being 96.9%, 90.7%, and 91.1% for EMA and anti-omega-gliadins IgG and IgA, respectively. Anti-tTG determination was performed on 103 samples (69 controls and 34 coeliac patients), finding 4 false results (2 false positive and 2 false negative), whereas anti-omega-gliadins showed 10 false results (5 false negative and 5 false positive), 3 of which were coincident with anti-tTG determination. To compare the reactivity of anti-gliadin antibodies, alpha-, beta-, gamma- and omega-gliadins were isolated under non-denaturing conditions by acid preparative electrophoresis and cation-exchange fast protein liquid chromatography (FPLC) and used in an indirect ELISA test. The composition of these fractions was analysed by means of capillary electrophoresis, showing no cross-contamination among them. The comparison of results using purified gliadins shows that omega-gliadins present a differential reactivity that has not previously been documented. Results using omega-gliadins isolated by either preparative electrophoresis or FPLC were similar. Tests using the purified omega-gliadin fraction present the best performance when anti-gliadin antibodies are evaluated.

Combination of various analytical techniques for speciation of low molecular weight aluminium complexes in plant sap

The possibilities for speciation of low molecular weight aluminium organic acid complexes (LMW-Al) in plant sap were investigated by applying a combination of various chromatographic and spectroscopic techniques. The behavior of different LMW-Al complexes was investigated in synthetic aqueous solutions on anion and cation exchange fast protein liquid chromatography (FPLC) columns with inductively coupled plasma – atomic emission spectrometry (ICP-AES) or electrothermal atomic absorption spectrometry (ETAAS) detection. Plant sap of Sempervivum tectorum with a high concentration of Al (9.3 μg cm–3) and Sansevieria trifasciata with an appreciably lower Al concentration (0.065 μg cm–3) were analyzed. The speciation study in plant sap indicated that the species present in these samples, existed as negatively charged LMW-Al complexes which were eluted at the retention time typical for Al-citrate and Al-aconitate. In order to identify Al binding ligands eluted under the chromatographic peak analysis by electrospray ionization (ESI)-MS technique using Z spray ion source were also applied. The predominant LMW-Al negatively charged complexes in Sempervivum tectorum and Sansevieria trifasciata were found to be Al-citrate and Al-aconitate. It was shown that other LMW organic acids do not form complexes with Al in the samples studied.

Speciation of trace amounts of aluminium in environmental samples by cation-exchange FPLC - ETAAS

Cation-exchange fast protein liquid chromatography - electrothermal atomic absorption spectrometry (FPLC - ETAAS) was applied for the speciation of trace amounts of aluminium. An aqueous 8 mol dm -3 NH4NO3 linear gradient elution in 10 min- utes enabled separation of positively charged monomeric aluminium species in aqueous solution. Separated aluminium species were collected in 0.5 cm3 fractions and aluminium determined off line after fourfold dilution by ETAAS. The ability of NH 4NO3 to completely decompose at low temperature in the graphite tube during the ashing step enabled quantitative and reproducible determination of aluminium. After applying Chelex-100 resin in a cleaning procedure of the eluent, the eluent blank was very low (< 1.0 ng cm-3 Al). When 1 cm3 of sample was injected on the column resin, the limit of detection (3 s) for the separated aluminium species determined by ETAAS was found to be 3 ng cm-3. Good repeatability of measurement of separated aluminium species (relative standard deviation ∠5%) was obtained under the recommended analytical conditions. The influence of some inor- ganic and organic complexing ligands at pH values of 4.0 and 6.0 on the speciation of aluminium was also investigated. The method developed was successfully applied for the speciation of aluminium in soil extracts and natural water samples at ng cm -3 levels.

Combination of three analytical techniques for speciation of Al in environmental samples

An analytical scheme is proposed which combines three speciation techniques for determination of particular Al species in soil extracts and percolating waters. A cation-exchange fast protein liquid chromatography — inductively coupled plasma atomic emission spectrometry (FPLC-ICP-AES) procedure, a microcolumn chelating ion-exchange chromatography- atomic absorption spectrometry (MCC-ETAAS) technique and the 8-hydroxyquinoline spectrophotometric method (8HQ-spectrophotometry) were employed. The FPLC-ICP-AES procedure offers determination of Al3+ (retention time 4.5 min) and Al(OH)2+ species (retention time 4.0 min) which are separated from Al(OH)+2 (retention time 1.5min). AlF2+ coelutes with Al(OH)2+ species, while Al(SO4)+, AlF+2 and negatively charged Al organic complexes coelute with Al(OH)+2 species. The MCC-ETAAS technique enables determination of the sum of positively charged monomeric aqua- and hydroxy-Al species plus sulphate- and fluoro-Al complexes. Employing the 8HQ-spectrophotometry the sum of positively charged monomeric aqua- and hydroxy-Al species plus sulphato- and most of the labile organic Al species are determined. The sensitivities of these selected techniques were adequate for speciation of Al in the samples analyzed. On the basis of the specific selectivity of a particular technique various groups of Al species may be determined. Thus, the comparison of analytical data from complementary procedures provides more comprehensive information on Al species present in soil extracts and percolating waters.

Study of the polymorphism of caprine milk caseins by capillary electrophoresis.

Polymorphism of caprine milk proteins was studied by capillary electrophoresis. Identification of casein (CN) fractions was effected by using isolated fractions from cation-exchange fast protein liquid chromatography. Genetic polymorphisms in caprine alpha s2-CN, alpha s1-CN, beta-CN and kappa-CN have been determined. kappa-CN A and B, beta-CN A and null, alpha s2-CN A, B and C, alpha s1-CN A, B, C and null, and other forms with intermediate and low alpha s1-CN content have been identified. The capillary electrophoresis method made it possible to analyse whole caprine milk using simple sample preparation and was rapid, automated and suitable for phenotyping studies. This method may also permit the quantitative study of different protein fractions.

Isolation and Amino Acid Sequences of Two Kunitz-Type Protease Inhibitors From the Sea Anemone Anthopleura aff. xanthogrammica

Two protease inhibitors (AXPI-I and -II) were isolated from the sea anemone Anthopleura aff. xanthogrammica by a combination of acetone precipitation, gel filtration on Sephadex G-75, cation-exchange fast protein liquid chromatography (FPLC) on Mono S and reverse-phase HPLC on TSKgel ODS-120T. Both inhibitors are basic polypeptides, and their amino acid compositions are characterized by the presence of six half-Cys residues and the absence of Met and Trp. They are potently active against trypsin; inhibition of other serine proteases (α-chymotrypsin and elastase) is also displayed by only AXPI-I. However, the inhibitors show no affinity for metallo-proteases and cysteine proteases. Analyses of the N-terminal portion and enzymatic fragments established their complete amino acid sequences comprising 58 residues. The overall sequence homology and the conserved location of all half-Cys residues confirmed that the A. aff. xanthogrammica inhibitors belong to the Kunitz-type family.

Speciation of aluminium in soil extracts by employing cation-exchange fast protein liquid chromatography–inductively coupled plasma atomic emission spectrometry

A fast protein liquid chromatographic–inductively coupled plasma (ICP) atomic emission spectrometric procedure was developed for the speciation of Al over a wide pH range, from acidic to alkaline regions. Positively charged monomeric Al species in synthetic aqueous solutions were separated from polymeric, neutral and negatively charged species on a cation-exchange fast protein LC column. Linear gradient elution with aqueous NaNO3(8 mol dm–3) at a flow rate of 1.0 cm3 min–1 over 10 min was employed for the separation. The separated Al species were determined ‘off-line’ by ICP atomic emission spectrometry in 0.5 cm3 fractions, which were diluted prior to analysis; yttrium was added as the internal standard. Retention times were found to be 1.5 min for Al(OH)2(H2O)4+[Al(OH)2+], 4.0 min for Al(OH)(H2O)52+[Al(OH)2+] and 4.5 min for Al(H2O)63+[Al3+]. Good reproducibility of measurement (relative standard deviation 1.5%) was obtained in the optimum concentration range. The limit of detection (3s) for the separated Al species was found to be 120 ng cm–3(1.0 cm3 sample). The distribution of Al species over a wide pH range agreed with the reported calculated data and, in general, agreed also with the data from the 8-hydroxyquinoline spectrophotometric method. An investigation of the effect of inorganic and organic complexing ligands on the speciation of Al indicated that positively charged [Al(SO)4+, AlF2+] and negatively charged (oxalate and citrate) Al species co-eluted with Al(OH)2+ and were separated from Al(OH)2+ and Al3+ species, while AlF2+ co-eluted with Al(OH)2+ species. At lower pH values (e.g., 4.0), aluminium citrate was partially co-eluted with Al3+ and Al(OH)2+ species. The same observations were found when soil extracts were analysed.

A Chromatographic Analysis of Capsid Protein Isolated from Alfalfa Mosaic Virus: Zinc Binding and Proteolysis Cause Distinct Charge Heterogeneity

The capsid protein (CP) of alfalfa mosaic virus (AIMV) is required for viral replication when susceptible plants are inoculated with purified vital genomic RNA. The discovery of AIMV CP in the zinc activated RNA-dependent RNA polymerase complex prompted our further investigation of AIMV virions and the potential involvement of AIMV CP in metal binding. AIMV CP, isolated from nucleoprotein components, fractionated into four distinct ionic species when purified by cation exchange fast protein liquid chromatography. The CP existed as zinc complexed homodimers, metal-free homodimers, and two forms of proteolyzed heterodimers, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography, amino-terminal sequencing, and atomic absorption spectroscopy. Although the relative amounts of proteolyzed heterodimers varied, the ratio of zinc complexed homodimers to metal-free homodimers (1:10) was constant between virus and protein isolations for the strains 425 and WISC14. Purified metal-free and zinc-complexed homodimers could be interconverted in vitro by incubation with zinc chloride or with the metal chelator, sodium diethyldithiocarbamate (NaDDC). The potential role of zinc in AIMV nucleoprotein structure and infectivity was investigated by treatment of the virions with NaDDC. Electron microscopy and sucrose density gradient studies failed to detect any gross structural changes for zinc depleted virus; however, a decrease in infectivity was observed with local lesion leaf assays, suggesting a functional role for zinc in viral replication.

Human growth factor for murine interleukin (IL)-9 responsive T cell lines: co-induction with IL-6 in fibroblasts and identification as LIF/HILDA.

Two mouse helper T cell clones that proliferate in response to murine interleukin (IL)-9 could also be grown in conditioned medium of stimulated human connective tissue cells. The activity was not due to known T cell growth factors including human IL-9, which is not effective on mouse cells. This growth-stimulatory activity for TS1 cells (GATS) was co-induced with IL-6 on normal fibroblasts and certain sarcoma cell lines stimulated with IL-1, double-stranded RNA, virus or phorbol ester. However, the conditions for optimal induction and the kinetics of production were found to be different for IL-6 and GATS. GATS from phorbol ester-stimulated human hepatosarcoma cells co-purified with IL-6, but could be separated from it by subsequent cation-exchange fast-protein liquid chromatography and reverse-phase high-performance liquid chromatography. Homogeneous tumor cell-derived GATS was a 25-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas IL-6 produced by these cells appeared in its 23-kDa form. Pure GATS was found to be inactive in the B cell hybridoma growth assay for IL-6. Finally, GATS was identified by NH2-terminal sequence analysis of the mature protein as leukemia inhibitory factor or human interleukin for DA cells (LIF/HILDA). The effect of LIF/HILDA on T cells was not mediated by IL-2, IL-4 or IL-9 production. Since this cytokine has not previously been reported to act on T cells, further investigation of its role in T cell activation should be taken into consideration.