Identification of the human 26-kD protein, interferon beta 2 (IFN-beta 2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor.

A factor that promotes the growth of certain B cell hybridomas and of plasmacytomas is shown to be produced by normal human fibroblasts and by a line of human osteosarcoma cells (MG-63) after treatment with IL-1 or TNF. The hybridoma-plasmacytoma growth factor (HPGF) is identified with a 26 kD protein whose mRNA was previously shown to be induced in the same cells by the same inducers. First, poly(A)-rich RNA extracted from IL-1-treated cells could be enriched in HPGF-mRNA content by hybridization to 26 kD cDNA. Second, MG-63-derived HPGF purified to electrophoretic homogeneity was subjected to amino acid sequence analysis, whereby the NH2-terminal sequence was found to match the nucleotide sequence of a 26 kD cDNA clone.

Cloning and characterization of a cDNA for a new mouse T cell growth factor (P40).

Recently, we described a murine helper T cell-derived molecule with T cell growth factor activity that is functionally and structurally distinct from IL-2, IL-4, and other known growth factors. This molecule, designated P40, was identified as a glycoprotein capable of supporting antigen-independent growth of certain helper T cell clones. Here, we report the cloning and expression of a cDNA for this new growth factor. The predicted mature protein is a cationic cysteine-rich polypeptide of 14 kD without significant homology to previously sequenced proteins.

Androgenicity and venous endothelial function in post-menopausal women.

BACKGROUND: Endothelial dysfunction is one of the early signs of cardiovascular damage. High androgen levels have been related to inflammatory endothelial markers in pre- and post-menopausal women. AIM: This cross-sectional study aimed at investigating whether free androgen index (FAI) [estimated by dividing total testosterone (nmol/l) by SHBG (nmol/l) x 100] is related to endothelial function during post-menopause. SUBJECTS AND METHODS: Twenty-six post-menopausal women were assessed with the dorsal hand vein compliance technique. Acetylcholine (Ach) and sodium nitroprusside (SNP) dose-response curves were constructed to test endothelium-dependent and independent relaxation, respectively. RESULTS: Mean age was 54 yr (+/-4) and median time since menopause was 6 yr (interquartile range: 3-9). Patients were stratified according to FAI levels into two groups: FAI greater than or less than the group median of 2.5. Waist-to-hip ratio (WHR) was significantly higher in the group with FAI>2.5, as well as median dose of Ach for maximal vasodilation [720 (360-3600) ng/min with FAI>2.5 vs 36 (0.36-360) ng/min with FAI

Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors.

The Lewis Y (Ley) antigen is a blood group-related antigen that is expressed in a high proportion of epithelial cancers (including breast, colon, ovary, and lung cancer) and is an attractive target for monoclonal antibody-directed therapy. The murine monoclonal 3S193 (IgG3) was generated in BALB/c mice by immunization with Ley-expressing cells of the MCF-7 breast carcinoma cell-line. The murine 3S193 showed high specificity for Ley in ELISA tests with synthetic Ley and Ley-containing glycoproteins and glycolipids and also reacted strongly in rosetting assays and cytotoxic tests with Ley-expressing cells. We generated a humanized form of the murine 3S193 antibody by linking cDNA sequences encoding the variable region of murine 3S913 with frameworks of the human KOL heavy chain and REI K chain. The genes for the humanized 3S193 monoclonal antibody IgG1 were transfected into mouse myeloma NS0 cells and cloned for the establishment of high antibody-producing colonies. Humanized 3S193 antibody was subsequently produced through in vitro culture and under good manufacturing practice conditions using hollow-fiber bioreactors. The purified humanized 3S193 (hu3S193) was subsequently characterized and validated for use in preliminary immunotherapy investigations. hu3S193 reacted specifically with Ley antigen, with similar avidity to the murine form. hu3S193 demonstrated potent immune effector function, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 microg/ml). The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7, Ley-positive cells. Six i.v. doses of up to 1 mg of hu3S193 were administered to animals bearing established tumors (120-130 mm3) with no significant effect on tumor growth. In contrast, in an MCF-7 xenograft preventive model, a 1-mg hu3S193 dosage schedule was able to significantly slow tumor growth compared with placebo and isotype-matched control IgG1 antibody. hu3S193 has promise for immunotherapy of Ley-positive tumors and is currently entering Phase I clinical trials.

Primary structure of ovine pituitary basic fibroblast growth factor.

The complete amino acid sequence of basic FGF (146 residues) from ovine pituitary glands has been established. This has been achieved by the sequence analysis of subnanomole amounts of the intact molecule and of peptides derived by enzymatic digestions with clostripain, chymotrypsin, pepsin and Staphylococcus aureus V8 protease. Microbore HPLC, employing 1-2 mm i.d. columns, was used to purify, concentrate and buffer-exchange the FGF peptides. A novel application of ion-pairing chromatography was employed to isolate peptides which were not retained on conventional reversed-phase systems. There is only one positional difference between the ovine and bovine basic FGFs, but there are 3 positional differences between ovine and human basic FGFS.

Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase.

The NH2-terminal amino acid sequence of the Mr 26 000 glutathione S-transferase (EC 2.5.1.18) of Schistosoma japonicum (Sj26) has been deduced by RNA and protein sequence analysis. Using this information, a bacterial plasmid has been constructed that directs the synthesis of the entire Sj26 molecule in Escherichia coli. Recombinant Sj26 exhibits glutathione S-transferase activity and can be readily purified from bacteria in a one-step procedure under non-denaturing conditions. The availability of recombinant Sj26 in essentially unlimited quantities will aid its assessment as a candidate vaccine molecule in schistosomiasis and could eventually lead to the rational design of a drug targetted on schistosome glutathione S-transferases.

The protein sequenator: a new extraction principle and a metal reaction cup.

A simpler and more efficient extraction procedure is proposed for solvent extraction of the Edman degradation procedure in the protein sequenator. The method is less likely than the conventional one to cause wash-out of peptides or proteins from the reaction cup while the very small solvent volumes required substantially decrease the running costs of the instrument. It has been used successfully (98% repetitive yield) with a metal reaction cup in the sequenator.

Recombinant NY-ESO-1 cancer antigen: production and purification under cGMP conditions.

The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.

Murine hybridoma/plasmacytoma growth factor. Complete amino-acid sequence and relation to human interleukin-6.

Murine interleukin-HP1 (HP1) was originally identified as a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas and plasmacytomas. This growth factor was recently shown to stimulate both normal B-cell differentiation and T-cell growth factor activity. We have determined the complete amino acid sequence of HP1 on 40 micrograms (approximately 2 nmol) protein using a combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC, peptide mapping and automated amino acid microsequence analysis. Ion-pairing chromatography was employed to isolate hydrophilic peptides which were not retained on conventional reversed-phase HPLC systems. The molecule consists of 187 amino acid residues with a calculated molecular mass of 21710 Da. Although there is virtually no similarity between the NH2-terminal region of HP1 and its human biological counterpart (26-kDa protein/interferon-beta 2 = B-cell stimulatory factor-2/interleukin-6), these studies demonstrate extensive amino acid similarity in the middle and COOH-terminal regions of these molecules suggesting that HP1 is the murine homologue of human interleukin-6.

The amino acid sequence of component 7c, a type II intermediate-filament protein from wool.

Component 7c is one of the four homologous type II intermediate-filament proteins that, by association with the complementary type I proteins, form the microfibrils or intermediate filaments in wool. Component 7c was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage reactions. It is an N-terminally blocked molecule of 491 residues and Mr (not including the blocking group) of 55,600; the nature of the blocking group has not been determined. The predicted secondary structure shows that component 7c conforms to the now accepted pattern for intermediate-filament proteins in having a central rod-like region of approximately 310 residues of coiled-coil alpha-helix flanked by non-helical N-and C-terminal regions. The central region is divided by three non-coiled-coil linking segments into four helical segments 1A, 1B, 2A and 2B. The N-and C-terminal non-helical segments are 109 and 71 residues respectively and are rich in cysteine. Details of procedures use in determining the sequence of component 7c have been deposited as a Supplementary Publication SUP 50152 (65 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257,5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 7c, peptides CN1, CN2 and CN3, and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, (3) details of methods used to determine the C-terminal sequence of peptide CN3, and (4) detailed evidence to justify a number of corrections to the previously published sequence.

Sequence interrelationships of the subunits of vicilin from pea seeds.

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than Mr∼50 000 (i.e., Mr 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within Mr∼50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than Mr∼50 000 arose by endoproteolytic cleavage of parent molecules within the Mr∼50 000 group. Cleavage in different Mr 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than Mr∼50 000, with the possible exception of the Mr34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the Mr∼50 000 group.

The amino-terminal sequence of the 85-90K nonhormone binding component of the molybdate-stabilized estradiol receptor from calf uterus.

The first six N-terminal amino acid residues of the 85-90K non-estrogen binding component of the calf uterine, molybdate-stabilized estradiol receptor have been determined by Edman degradation. After affinity chromatography of the stabilized receptor oligomer, the 85-90K unit was purified to homogeneity by preparative gel electrophoresis using electroelution for protein recovery. Inverse-gradient high performance liquid chromatography provided the 85-90K protein suitable for amino-terminal sequence analysis.

Complete amino acid sequence of a new murine T-cell growth factor P40.

A new murine T-cell growth factor, designated P40, which supports growth of helper T-cells in the absence of interleukin-2, interleukin-4 and antigen has been isolated from helper T-cell lines in sufficient quantities (100 micrograms) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed-phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N-terminal sequence data on P40 were unsuccessful due to N-terminal blockage of the native molecule. The nature of this N-terminal blocking was established using a combination of amino acid analysis, fast-atom-bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T-cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict four N-linked glycosylation sites in the P40 molecule.

Purification and partial amino acid sequence of annexin V from porcine gastric mucosal membranes.

1. Annexin V has been purified from Triton X-100 extracts of porcine gastric mucosal membranes by a combination of chromatography on concanavalin A-Sepharose and DEAE-Sepharose, and preparative gel electrophoresis. 2. No N-terminal amino acid sequence was detected. 3. The sequences of 11 tryptic peptides were determined, amounting to a total of 121 amino acids, or 38% of the molecule. 4. When the peptides were compared with the cDNA-derived sequence of human annexin V, only three substitutions were observed. 5. Human and porcine annexin V are 97% homologous within the sequenced regions.

Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas.

A T-cell-derived lymphokine was identified by its ability to support the growth of a subset of B-cell hybridomas. Hybrids that failed to survive in the absence of this molecule represented a major proportion of rat-mouse hybridomas but were very rare among mouse-mouse B-cell hybrids. Stable factor-dependent B-cell hybridomas were used to monitor the purification of the growth factor from the supernatant of a clonotypically stimulated mouse helper T-cell clone. Sequential fractionation using gel filtration, anion-exchange chromatography, and reversed-phase HPLC resolved the factor from other B-cell growth factors and yielded a single-chain protein characterized by a major charge (pI = 5-7) and molecular mass (22- to 29-kDa) heterogeneity, probably due to variations in glycosylation. The NH2-terminal amino acid sequence of this protein, which is active on B-cell hybridomas in the 0.1 pM range, showed no significant homology with that of known lymphokines. Because the purified factor also supported the growth and survival in vitro of murine plasmacytomas (to be published elsewhere), it was provisionally designated interleukin-HP1 (where H stands for hybridoma and P stands for plasmacytoma).

Isolation and partial amino acid sequence of a 78 kDa porcine gastrin-binding protein.

1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle15]-gastrin. 2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides. 3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55-61). The remaining 7 peptides were derived from the beta-subunit of the gastric H+/K(+)-ATPase. 4. The gastrin-binding activity remained in association with p78, and could be separated from the beta-subunit of the gastric H+/K(+)-ATPase, during chromatography on tomato lectin-Sepharose. 5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.

Structural studies on a 75-kDa glycoprotein isolated from porcine gastric mucosal membranes: close homology with the 78-kDa glucose-regulated family of proteins.

A 75-kDa glycoprotein (P75) has been purified to homogeneity from washed membranes isolated from the corpus of porcine gastric mucosa. The purification procedure employed chromatography on concanavalin A-Sepharose and DEAE-Sepharose, and preparative polyacrylamide gel electrophoresis. Reversed-phase microbore high-performance liquid chromatography was employed to fractionate and purify a number of tryptic peptides generated from approximately 1100 pmol purified P75. The use of reversed-phase microbore (2.1 mm internal diameter) columns facilitated the purification of subnanomole amounts of polypeptides in small volumes (40-60 microliter) suitable for loading onto the gas-phase sequencer without further concentration. N-Terminal amino acid sequence analyses were performed on the intact polypeptide and on 13 tryptic peptides and one Staphylococcus protease V8 peptide, yielding 170 unique assignments; this corresponds to approximately 26% of the molecule. A comparison of this amino acid sequence information with the cDNA-deduced primary structure of a 70-kDa heat-shock-related protein (P72), which is expressed in normal rat liver reveals that these protein sequences are almost identical, differing in only 1 of the 170 positions positively assigned thus far. The probable correspondence of P72 with the 78-kDa glucose-regulated protein (GRP78) isolated from hamster fibroblasts has been reported.

Structural characterization of a murine myeloid leukaemia inhibitory factor.

A leukaemia inhibitory factor (LIF) which induces macrophage differentiation in M1 murine myeloid leukaemia cells and suppresses their proliferation in vitro has been isolated in sufficient quantities (30 micrograms) from Krebs ascites tumour cell conditioned medium to permit its partial characterization by amino acid sequence analysis. The combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC technology and microsequence analysis has enabled the positive identification of 125 of the total 179 amino acid residues (70%) in the molecule. The amino acid sequence data reported here permitted the isolation of a partial cDNA clone encoding LIF [Gearing et al. (1987) EMBO J. 6, 3995-4002]. A candidate C-terminus of the LIF molecule predicted from the amino acid sequence was confirmed by subsequent isolation of a cDNA clone corresponding to the C-terminus of the protein. No strong similarity was revealed when the amino acid sequence of LIF was compared with other haemopoietic growth factors, in particular granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and tumour necrosis factor-alpha or interleukins. The protein sequence data reported here indicate three sites of post-translational modification (N-linked glycosylation).

Purification and partial amino acid sequence of human aconitase.

Aconitase has been purified from membranes prepared from both the human gastric carcinoma cell line Okajima and from porcine gastric mucosa by chromatography on concanavalin A-Sepharose and carboxymethyl-Sepharose, and preparative polyacrylamide gel electrophoresis. Automated Edman degradation of the intact proteins yielded no N-terminal amino acid sequence due, presumably, to N-terminal blockage. Sequence analysis of tryptic peptides derived from S-carboxymethyl porcine and human aconitases established the positions of 95 and 64 amino acid residues, respectively. The amino acid sequence data for porcine aconitase was in perfect agreement with the previously reported cDNA-deduced amino acid sequence [Zheng et al. (1990) J Biol Chem 265:2814-2821]. Comparison of the human amino acid sequence data with the cDNA-deduced amino acid sequence of porcine aconitase indicated that these two proteins have 95% amino acid sequence identity within the sequenced region.

Rendimiento académico y conductas antisociales y delictivas en alumnos de Educación Secundaria Obligatoria / Academic Achievement and Antisocial Behavior in Public Secondary Education Students

The objective of this study was to analyze the relationship between violence and academic achievement, two of the most important problems in Secondary Education in Spain. Participants were 881 students 14-18 years old (M= 15.20, SD= 0.901) from 6 public centers of Secondary Education. Results indicate that men show more prevalence in antisocial behavior, increasing with age and school year. Failure in academic achievement was associate to antisocial and delinquent behaviors. Also, the educational level of mothers was related to the presence of antisocial behavior (AU)

El objetivo de este estudio fue analizar la influencia del rendimiento académico y del nivel educativo de padres/madres sobre la presencia de conductas antisociales y delictivas en el alumnado, dos problemas graves en Educación Secundaria Obligatoria en España. Partici- paron en el estudio 881 alumnos con edades entre 14 y 18 años (M= 15,20; DT= 0,901) de seis centros públicos de Educación Secundaria Obligatoria. Los resultados indicaron que los hombres realizan mayor número de conductas antisociales y delictivas, aumentando su prevalencia con la edad y el curso escolar. El fracaso en el rendimiento académico también se asoció a la presencia de conductas antisociales y delictivas. Igualmente, el nivel de estudios de la madre/tutora se relacionó con la presencia de conductas antisociales (AU)