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Background:Despite increased neuronal death, senile plaques, and neurofibrillary tangles observed in patients suffering from Alzheimer’s disease (AD), the detailed mechanism of cell death in AD is still poorly understood.Method:We hypothesized that p38 kinase activates and then phosphorylates Bax, leading to its translocation to mitochondria in AD brains compared to controls. The aim of this study was to investigate the role of p38 kinase in phosphorylation and sub-cellular localization of pro-apoptotic Bax in the frontal cortex of the brains from AD and control subjects. Increased oxidative stress in AD individuals compared to control was evaluated by measuring the levels of carbonylated proteins and oxidized peroxiredoxin, an antioxidant enzyme. The relative amounts of p38 kinase and phospho-Bax in mitochondria in AD brains and controls were determined by immunoblot analysis using the respective antibody against each protein following immunoprecipitation.Results:Our results showed that the levels of oxidized peroxiredoxin-SO3and carbonylated proteins are significantly elevated in AD brains compared to controls, demonstrating the increased oxidative stress.Conclusion:The amount of phospho-p38 kinase is increased in AD brains and the activated p38 kinase appears to phosphorylate Thr residue(s) of Bax, which leads to its mitochondrial translocation, contributing to apoptosis and ultimately, neurodegeneration.

A model is presented that is applicable to a wide range of peak-shaped voltammetric signals. It may be used, via curve-fitting, to resolve severely overlapped peaks, irrespective of the degree(s) of reversibility of the electrode processes. The resolution procedure has been thoroughly tested for several voltammetric and polarographic techniques (differential pulse, square wave and pseudo-derivative normal pulse), using reversible, quasireversible and irreversible electrochemical systems.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.

The global method for analyzing electrochemical processes controlled by diffusion and by the kinetics of heterogeneous electron transfer is redeveloped or application to electrodes haveing a spherical geometry. Heretofore, the method has been restricted to experiments in which plannar diffusion is the sole mode of transport to the electrode surface. The global method is based on a three-dimensional concept and has the advantage of using all of the data available from a voltammogram to measure the heterogeneous charge-transfer rate constant, the reversible halfwave potential, the diffusion coefficient, and the charge-transfer coefficient. Cyclic voltammetry of 2-methyl-2-nitropropane reduction at a mercury sphere is reported. The parameters measured by spherical global analysis are concordant and in excellent agreement with literature values.

A novel type of whole inactivated simian immunodeficiency virus (SIV) virion vaccine immunogen with functional envelope glycoproteins was evaluated, without adjuvant, in rhesus macaques. Immunogens included purified inactivated virions of SIVmac239, a designed mutant of SIVmac239 with gp120 carbohydrate attachment sites deleted (SIVmac239 g4,5), and SIVmneE11S. The vaccines were noninfectious, safe, and immunogenic, inducing antibody responses and cellular responses, including responses by CD8+ lymphocytes. Interpretation of protective efficacy following intrarectal challenge was complicated by incomplete take of the challenge in some SIV naïve controls.

A simian immunodeficiency virus (SIV)(Mne) DNA clone was constructed that produces viruses containing a four amino acid deletion in the second zinc finger of the nucleocapsid (NC) domain of the Gag polyprotein. Viruses produced from this clone, although non-infectious both in vitro and in vivo, complete a majority of the steps in a single retroviral infection cycle. Eight pig-tailed macaques (Macaca nemestrina) were inoculated intramuscularly and subcutaneously three times over the course of 24 weeks with the NC mutant expressing DNA. These macaques, and four controls, were then challenged mucosally (intrarectally) with the homologous virus (SIV Mne CL E11S) and monitored for evidence of infection and clinical disease. Prior to challenge, a measurable humoral immune response was noted in four of eight immunized macaques. After challenge, all 12 macaques became infected, although four immunized animals greatly restricted their viral replication, and one immunized animal that controlled replication remains antibody negative. No disease has been evidence during the 46-week period of monitoring after challenge.

HIV-1 nucleocapsid protein (NCp7) is a double zinc-fingered protein that has been traditionally implicated in viral RNA recognition and packaging, in addition to its tight association with genomic RNA and tRNA primer within the virion nucleocapsid. The availability of large quantities of viral or recombinant wild-type NCp7 and mutant p7 has made possible the assignment of the different roles that structural motifs within the protein play during RNA binding. At low ionic strength binding to the homopolymeric fluorescent RNA, poly(epsilonA), is electrostatically driven and four sodium ions are displaced. Arg7 in the flanking N-terminal region, Lys20 and Lys26 in the first zinc finger and one positively charged residue (attributed to Lys41) in the second zinc finger are involved in electrostatic contacts with RNA. The p7 zinc fingers do not function independently but concomitantly. The first zinc finger (both isolated or in the context of the full-length protein) has a more prominent electrostatic interaction than the second one. The second zinc finger dominates the non-electrostatic stabilization of the binding to RNA due to stacking of its Trp residue with nucleic acid bases. Mutations in the highly conserved retroviral Zn-coordinating residues (CCHC) to steroid hormone receptor (CCCC) or transcription factor (CCHH) metal cluster types do not affect RNA binding. In spite of the limited impact in RNA binding affinity in vitro or RNA packaging in vivo that such mutations or structural alterations impart, they impair or abolish virus infectivity. It is likely that such an effect stems from the involvement of NCp7 in crucial steps of the virus life cycle other than RNA binding.

Although most viral vaccines used in humans have been composed of live attenuated viruses or whole killed viral particles, the latter approach has received little attention in research on experimental primate immunodeficiency virus vaccines. Inactivation procedures involving heat or formalin appear to adversely affect the viral envelope proteins. Recently we have inactivated human immunodeficiency virus type 1 (HIV-1) with the compound 2,2'-dithiodipyridine (Aldrithiol-2, Aldrich, Milwaukee, WI), which inactivates infectivity of retroviruses by covalently modifying the nucleocapsid zinc finger motifs. HIV-1 inactivated with Aldrithiol-2 retained the conformational and functional integrity of the viral and virion-associated cellular proteins on the viral membrane. We have extended our studies of zinc finger targeted inactivation to simian immunodeficiency virus (SIV) and evaluated the feasibility of applying the procedures to large scale (30 l) production and purification of the primate immunodeficiency viruses. There was no detectable residual infectivity of SIV after treatment with 1 mM Aldrithiol-2 (5 logs inactivation). Treatment with Aldrithiol-2 resulted in extensive reaction with the nucleocapsid protein of treated virus, as shown by immunoblot and high-performance liquid chromatography (HPLC) analysis. As expected, the virion gp120SU appeared to be completely unreactive with Aldrithiol-2. Sucrose gradient purification and concentration procedures resulted in little loss of viral infectivity or virion-associated gp120SU. When tested in a gp120-CD4 dependent cell binding assay, the inactivated virus bound to cells comparably to the untreated virus. Analysis of gp120-CD4 mediated postbinding fusion events showed that the inactivated virus could induce CD4-dependent fusion with efficiencies similar to the untreated virus. Inactivation and processing of primate immunodeficiency viruses by methods described here results in highly concentrated virus preparations that retain their envelope proteins in a native configuration. These inactivated virus preparations should be useful in whole killed-particle vaccine experiments as well as laboratory reagents to prepare antisera, including monoclonal antibodies, and to study noninfective virion-cell interactions.

We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.

All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two zinc fingers, consisting of the sequence C-X2-C-X4-H-X4-C. Rice et al. (Science 270:1194-1197, 1995) have described a series of compounds which inactivate HIV-1 particles and oxidize the sulfur atoms in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. In contrast, the compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV-1 by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses, and the lethality of all known mutations altering the zinc-binding residues, suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.

The aim of this study was to assess the demand for warfarin prescribing and monitoring, and to identify patients with atrial fibrillation who might benefit from warfarin therapy. The study was carried out in the population of Tayside, Scotland (400,000 people) using patient-specific dispensed prescribing and hospitalisation data from the Medicines Monitoring Unit at the University of Dundee.The incidence and prevalence of digoxin and warfarin prescribing were calculated between 1989 and 1993. Patients dispensed digoxin in 1993 were assumed to have atrial fibrillation and they were stratified into high risk groups for an adverse thromboembolic event based on past medical history. The numbers of patients at high risk who were judged to be possible candidates for warfarin were calculated.The prevalence of warfarin prescribing is increasing in Tayside and is mainly for elderly patients. There were also many patients assumed to have atrial fibrillation who were at particularly high risk for an adverse thromboembolic event, who had no record of warfarin prescribing. Only 35% received warfarin. Even given the methodological limitations of this study, and the use of aspirin as an alternative prophylactic agent, it is likely that these patients have been a source of increased prevalence of warfarin prescribing since 1993 and will be in the future. Other indications for warfarin prescribing are also increasing.It is anticipated that there will be increasing demands for anticoagulant monitoring, which will need to be met either by increasing the capacity of existing clinics, or by increasing the role of primary care.

Oxidants targeted toward inactivation of the nucleocapsid zinc finger protein are under development as antiviral agents, especially for use against human immunodeficiency virus. In the present study, electrospray ionization-mass spectrometry is used to follow in situ the progress of the reactions of 2,2'-dithiodipyridine and disulfiram with recombinant nucleocapsid protein p7 (Ncp7) from human immunodeficiency virus-1 at pH 7.4. Both reagents react with the two zinc fingers in the protein, resulting in the ejection of two zinc ions and the formation of oxidized apo-Ncp7 with three intramolecular disulfide bonds. The ejection of zinc by 2,2'-dithiodipyridine occurs in two steps. Alkylation of unreacted cysteine residues with N-ethylmaleimide after a 2-min reaction with 2,2'-dithiodipyridine reveals that the carboxyl-terminal zinc finger is disrupted first. Cys-49, Cys-36, and, to a lesser extent, Cys-39 are all shown to be target residues for initial electrophilic attack. In the reaction of disulfiram with Ncp7, ejection of the two zinc ions also occurs in two steps; however, the fully oxidized apo-Ncp7 is formed more rapidly. Thus, after a 40-min reaction, 45% of native Ncp7 is oxidized by 2,2'-dithiodipyridine, whereas 75% is oxidized by disulfiram.

We have biochemically purified a 27-kDa serine protease (designated RNK-Tryp-2) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) which has tryptase activity. Utilizing molecular sieve chromatography and reverse-phase HPLC, we purified RNK-Tryp-2 to homogeneity and sequenced 33 NH2-terminal amino acids. Oligonucleotide primers were used in the PCR to generate a 528-bp cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate an 884-bp RNK-Tryp-2 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 233 amino acids which does not have potential sites for N-linked glycosylation. The cDNA encodes a leader peptide of at least 25 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the N-terminus, and the His, Asp, and Ser amino acids that form the catalytic triad of serine proteases, are conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Tryp-2 is distinct protease. Southern blot analysis suggests the existence of one or more related genes. A single 1.3-kb mRNA transcript was detected by Northern blot analysis of total cellular RNA from the in vivo passaged RNK-16, rat splenocytes, lung and liver nonparenchymal cells, as well as in highly purified rat LGL and T cells. RNK-Tryp-2 is a novel serine protease that is expressed in the granules of large granular lymphocytes.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.

We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.

Single-cell clones of HIV-1 (FRE-3) or SIV/Mne infected HuT 78 cells were obtained by plating dilutions of virally infected HuT 78 cells on a monolayer of sheep choroid plexus cells in 96-well microtiter plates. Several of these clones produce HIV-1 virus mutants that accumulate the gag precursor polyprotein and lack a functional protease. These protease-deficient viruses are non-infectious and consist of aberrant "immature" virus particles as determined by electron microscopy. Several SIV mutants are also described that produce large amounts of either the envelope glycoprotein gp120 or the nucleic acid binding gag protein. These mutants are useful for the purification of these retroviral proteins, in developing assays of protease inhibitors, and in preparing SIV envelope protein vaccines.

During retroviral maturation gag precursors are proteolytically cleaved to mature gag proteins. The number of mature gag proteins and their order in the gag precursors of HIV-I3B, SIVMne (captive macaque isolate), and SIVCat (wild mangabey isolate) has been determined by N-terminal amino acid sequence analysis of mature gag proteins and alignment with predicted sequences of homologous gag precursors. For HIV-1 and SIVs maturation proteolysis results in six gag proteins and the gag precursor cleavage pattern is distinctive and different from cleavage patterns for all other known retroviruses.

We have studied the interactions of single-stranded polyribonucleotides with murine leukemia virus structural proteins p10, p10' (a p10 variant), and Pr65gag, as well as Rous sarcoma virus (RSV) pp12 (a p10 analog). Two quantitative assays have been used to monitor protein-RNA association: the fluorescence enhancement of polyethenoadenylic acid) poly(epsilon A) upon binding protein, and tryptophan fluorescence quenching upon binding to poly(U). With each assay p10 was shown to bind stoichiometrically to single-stranded RNA, covering a length of nucleic acid chain (occluded site size, n) of about 6 residues. RSV pp12 was also shown to bind to poly(epsilon A), with n = 5 +/- 1. Addition of NaCl to fully titrated MuLV p10-nucleic acid mixtures effected nearly complete restoration of poly(epsilon A) or MuLV p10 fluorescence. Under conditions of 0.06 M NaCl, p10 bound noncooperatively to poly(epsilon A) with an intrinsic association constant, K = 2.3 X 10(6) M-1. K and n determined in this study were shown to relate to Kapp determined by other methods, by the approximation Kapp approximately NK, where N is the number of binding sites along the polynucleotide chain ((nucleotides/chain)/n). Chemical modifications of the p10 cysteine residues did not alter the affinity for poly(epsilon A). The affinity of Pr65gag for poly(epsilon A) appears to be higher than that of p10.

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.

A low molecular weight polypeptide with multiplication-stimulating activity (MSA) was isolated from serum-free media conditioned by a clone of normal Buffalo rat liver cells. The purification of MSA was achieved by gel permeation chromatography of the acid-soluble growth-promoting activity on Bio-Gel P-10 in 1 M acetic acid and followed by reversed phase high pressure liquid chromatography on muBondapak C18 support using a linear gradient of acetonitrile in 0.05% trifluoroacetic acid. The primary structure of MSA has been determined. MSA is a single chain polypeptide of 67 residues, with a calculated molecular weight of 7,484, and displays 93% homology with the functionally related human insulin-like growth factor II (IGF-II). A comparison between the sequences of rat MSA and human IGF-II reveals only five amino acid substitutions. Based on the extensive amino acid sequence homology, we propose the term rat IGF-II for this newly isolated polypeptide.

Rasheed rat sarcoma virus, derived by in vitro cocultivation of two rat cell lines (Rasheed et al., Proc. Natl. Acad. Sci. U.S.A. 75:2972-2976, 1978), has been reported to code for a protein of 29,000 Mr, immunologically related to the 21,000 Mr src gene product of Harvey and Kirsten sarcoma viruses. Rat sarcoma virus p29 was thought to contain at least part of a rat type C virus structural protein, since antiserum prepared against whole rat virus was able to immunoprecipitate rat sarcoma virus p29 but not Harvey or Kirsten sarcoma virus p21 (Young et al., Proc. Natl. Acad. Sci. U.S.A. 76:3523-3527, 1979). We now report that antiserum directed against rat type C virus p15, but not viral p12, p10, or p27, immunoprecipitated rat sarcoma virus p29. The p15 antiserum was also able to immunoprecipitate both denatured p29 and a peptide derived by V-8 protease cleavage of p29, indicating that this antiserum contains antibodies directed against primary amino acid determinants. Finally, five separate isolates of rat sarcoma virus were found to code for p29, which indicates that a highly specific site of recombination is involved in the generation of sarcoma viruses in rat cells.

Interleukin 2 (IL 2) was purified from the conditioned medium of a gibbon T cell line, MLA144, which releases IL 2 constitutively. The IL 2 was obtained free of contaminating proteins by a simple process consisting of an initial batch purification on trimethylsilyl-controlled pore glass followed by reversed phase high pressure liquid chromatography. Overall recovery of IL 2 activity ranged from 70 to 100% of initial activity and yielded 2 X 10(6) or greater units of IL 2 per 15 liters of serum-free MLA144 conditioned medium. The specific activity of purified IL 2 ranged from 0.5 to 1 X 10(8) IL 2 U/mg protein. The purified IL 2 showed four molecular species when analyzed by two-dimensional isoelectric focusing-SDS-polyacrylamide gel electrophoresis. Each of the four molecular forms was active in the bioassay for IL 2 activity. Three molecular forms had apparent m.w. of 16,000 but different isoelectric points of 5.9, 6.3, and 6.7. One molecular form had an apparent m.w. of 15,000 and an isoelectric point of 7.2. The most abundant form of IL 2 had an apparent m.w. of 16,000 and an isoelectric point of 6.3. The purified IL 2 supported the growth of IL 2-dependent lymphocytes to a greater extent than did the same level of crude IL 2-containing MLA144 conditioned medium. The ability to purify large amounts of IL 2 by a rapid and efficient procedure will be of great help in both biochemical and immunologic studies of this lymphokine.

Considerations sur la theorie de la voltammetrie cyclique sur des microelectrodes ayant une epaisseur inferieure a 1 μm, dans le cas de processus aux electrodes ou l'etape du transfert d'electrons controle la cinetique. Exemple dans le cas de l'oxydation a un electron du ferrocene en cation ferrocenium

Highly enriched populations of human large granular lymphocytes (LGL), natural killer (NK) cells, and T cells were obtained from low and high density fractions, respectively, of discontinuous Percoll gradients. The NK cells were composed of 75 to 90% LGL, with the majority of the contaminating cells being monocytes. The T cells were greater than 95% OKT3+. The proliferative and cytotoxic progenitors in both fractions were examined by using a limiting dilution assay with interleukin 2 (IL 2) from four sources: 1) crude supernatant of a gibbon lymphoma (MLA-144), 2) purified (150,000-fold) MLA-144 IL 2, 3) partially purified human IL 2, and 4) purified recombinant human IL 2. The proliferative capacity was measured at day 7 by [3H]thymidine incorporation, whereas the progenitors of cells with NK-like activity were evaluated by assessing cytotoxic activity against K562 cells at day 8 in a 4-hr 51Cr-release assay. The frequency of proliferative progenitors among T cells was approximately 1/5 and was approximately 1/60 with LGL. Titration of the highly purified IL 2 preparation demonstrated that LGL proliferated with as little as 2 U of IL 2. The frequency of detectable cytotoxic progenitors in the LGL population, however, fell sharply when less than 40 U of IL 2 were employed. The T cells failed to demonstrate cytotoxic activity against the NK-susceptible target cells at any concentration of IL 2 tested. The IL 2 preparations also were examined for their ability to directly and rapidly enhance the cytotoxic activity of highly purified NK cells. All four preparations of IL 2 enhanced the cytotoxic activity of LGL without any detectable accessory requirement after incubation for as little as 6 hr, even though the MLA-144 IL 2 preparations were devoid of detectable interferons (IFN). These data indicate that IL 2 has dual effects on NK cells, regulating their activity was well as promoting their proliferation. Collectively, these results demonstrate that highly purified IL 2, devoid of other detectable lymphokines, is capable of supporting the growth of human NK cells and augmenting their in vitro activity. In parallel experiments, these same IL 2 preparations were quite active in causing the proliferation of T lymphocytes, clearly demonstrating a role of IL 2 in promoting the proliferation of NK cells as well as T cells. The mechanism of IL 2 boosting appears to be a direct interaction with LGL, resulting in the production of IFN gamma.(ABSTRACT TRUNCATED AT 400 WORDS)

A low molecular weight polypeptide with multiplication-stimulating activity (MSA) was isolated from serum-free media conditioned by a clone of normal Buffalo rat liver cells. The purification of MSA was achieved by gel permeation chromatography of the acid-soluble growth-promoting activity on Bio-Gel P-10 in 1 M acetic acid and followed by reversed phase high pressure liquid chromatography on muBondapak C18 support using a linear gradient of acetonitrile in 0.05% trifluoroacetic acid. The primary structure of MSA has been determined. MSA is a single chain polypeptide of 67 residues, with a calculated molecular weight of 7,484, and displays 93% homology with the functionally related human insulin-like growth factor II (IGF-II). A comparison between the sequences of rat MSA and human IGF-II reveals only five amino acid substitutions. Based on the extensive amino acid sequence homology, we propose the term rat IGF-II for this newly isolated polypeptide.

Urinary protein obtained from a patient with a highly malignant brain tumor (astrocytoma, grade IV) was adsorbed to trimethylsilyl controlled-pore glass beads and selectively eluted with acetonitrile to yield a high molecular weight (HMW) human transforming growth factor (hTGF). This HMW hTGF promoted clonogenic cell growth in soft agar and competed for membrane receptors with mouse epidermal growth factor. After surgical resection of the tumor, no HMW hTGF was found in urine. HMW hTGF generated a human EGF (hEGF) radioimmunoassay competitive binding curve similar to that of hEGF and parallel to that of a highly purified HMW form of hEGF previously reported to be present in trace concentrations in normal human urine. Both hEGF and HMW hEGF were clonogenic in soft agar, and their clonogenic activity as well as that of HMW hTGF was inhibited by anti-hEGF serum. Both HMW hTGF and HMW hEGF had 20 to 25% of the radioreceptor binding activity of hEGF. HMW hTGF purified from the pooled urine of several patients with malignant astrocytomas and HMW hEGF purified from normal control urine comigrated at Mr 33,000. Thus, HMW hTGF was indistinguishable from HMW hEGF in terms of apparent molecular size, epidermal growth factor receptor binding activity, epidermal growth factor immunoreactivity, and clonogenic activity. Urinary HMW hEGF/hTGF may be of tumor cell origin or may represent a response of normal host tissues to the tumor or its products.

Simian immunodeficiency virus (SIV)/Mne has been inoculated into three species of macaques and into baboons. Virus was isolated from all the macaques who subsequently died at 15 to 120 weeks (mean 80 weeks) with various manifestations of immune deficiency. Individual animals varied in their viral antibody profile as a function of time after infection. Independent SIV isolates obtained from African green monkeys and magabeys were compared to SIV/Mne for their ability to replicate in lymphocytes and macrophages and with respect to the immunological relatedness of their viral proteins. Antibodies present in human immunodeficiency virus-2 (HIV-2)-infected individuals were readily detected by the virus produced by a single-cell clone of SIV/Mne.

Interleukin 3 (IL 3) was initially defined as a factor in conditioned media from concanavalin A-stimulated lymphocytes (Con A CM) that induces the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of nu/nu splenic lymphocytes. To determine the spectrum of additional "biologic" activities, IL 3 was purified to homogeneity and its properties were assessed. The protein preparation was judged to be homogeneous IL 3 by the following criteria: 1) elution of a peak of IL 3 with a constant specific activity in the last step of purification, 2) presence of a single protein by SDS-PAGE analysis, 3) receptor-binding activity against IL 3-dependent cell lines, 4) a specific activity of congruent to 0.2 ng/ml required for 50% of maximal biologic activity, and 5) the presence of a single amino terminal sequence. With the use of this preparation of IL 3, the dose-response curves for 20 alpha SDH induction were identical or similar to the dose-response curves for the activities of 1) WEHI-3 growth factor, 2) mast cell growth factor, 3) P cell-stimulating factor, and 4) histamine-producing cell-stimulating factor. In addition, homogeneous IL 3 had colony-stimulating factor activity, although only approximately 2% of the total CSF activity found in Con A CM was associated with IL 3. The major peak of CSF activity could be resolved from IL 3 by DEAE column chromatography and lacked many of the biologic activities associated with IL 3.

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.

We have studied the interactions of single-stranded polyribonucleotides with murine leukemia virus structural proteins p10, p10' (a p10 variant), and Pr65gag, as well as Rous sarcoma virus (RSV) pp12 (a p10 analog). Two quantitative assays have been used to monitor protein-RNA association: the fluorescence enhancement of polyethenoadenylic acid) poly(epsilon A) upon binding protein, and tryptophan fluorescence quenching upon binding to poly(U). With each assay p10 was shown to bind stoichiometrically to single-stranded RNA, covering a length of nucleic acid chain (occluded site size, n) of about 6 residues. RSV pp12 was also shown to bind to poly(epsilon A), with n = 5 +/- 1. Addition of NaCl to fully titrated MuLV p10-nucleic acid mixtures effected nearly complete restoration of poly(epsilon A) or MuLV p10 fluorescence. Under conditions of 0.06 M NaCl, p10 bound noncooperatively to poly(epsilon A) with an intrinsic association constant, K = 2.3 X 10(6) M-1. K and n determined in this study were shown to relate to Kapp determined by other methods, by the approximation Kapp approximately NK, where N is the number of binding sites along the polynucleotide chain ((nucleotides/chain)/n). Chemical modifications of the p10 cysteine residues did not alter the affinity for poly(epsilon A). The affinity of Pr65gag for poly(epsilon A) appears to be higher than that of p10.

Highly enriched populations of human large granular lymphocytes (LGL), natural killer (NK) cells, and T cells were obtained from low and high density fractions, respectively, of discontinuous Percoll gradients. The NK cells were composed of 75 to 90% LGL, with the majority of the contaminating cells being monocytes. The T cells were greater than 95% OKT3+. The proliferative and cytotoxic progenitors in both fractions were examined by using a limiting dilution assay with interleukin 2 (IL 2) from four sources: 1) crude supernatant of a gibbon lymphoma (MLA-144), 2) purified (150,000-fold) MLA-144 IL 2, 3) partially purified human IL 2, and 4) purified recombinant human IL 2. The proliferative capacity was measured at day 7 by [3H]thymidine incorporation, whereas the progenitors of cells with NK-like activity were evaluated by assessing cytotoxic activity against K562 cells at day 8 in a 4-hr 51Cr-release assay. The frequency of proliferative progenitors among T cells was approximately 1/5 and was approximately 1/60 with LGL. Titration of the highly purified IL 2 preparation demonstrated that LGL proliferated with as little as 2 U of IL 2. The frequency of detectable cytotoxic progenitors in the LGL population, however, fell sharply when less than 40 U of IL 2 were employed. The T cells failed to demonstrate cytotoxic activity against the NK-susceptible target cells at any concentration of IL 2 tested. The IL 2 preparations also were examined for their ability to directly and rapidly enhance the cytotoxic activity of highly purified NK cells. All four preparations of IL 2 enhanced the cytotoxic activity of LGL without any detectable accessory requirement after incubation for as little as 6 hr, even though the MLA-144 IL 2 preparations were devoid of detectable interferons (IFN). These data indicate that IL 2 has dual effects on NK cells, regulating their activity was well as promoting their proliferation. Collectively, these results demonstrate that highly purified IL 2, devoid of other detectable lymphokines, is capable of supporting the growth of human NK cells and augmenting their in vitro activity. In parallel experiments, these same IL 2 preparations were quite active in causing the proliferation of T lymphocytes, clearly demonstrating a role of IL 2 in promoting the proliferation of NK cells as well as T cells. The mechanism of IL 2 boosting appears to be a direct interaction with LGL, resulting in the production of IFN gamma.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 2 (IL 2) was purified from the conditioned medium of a gibbon T cell line, MLA144, which releases IL 2 constitutively. The IL 2 was obtained free of contaminating proteins by a simple process consisting of an initial batch purification on trimethylsilyl-controlled pore glass followed by reversed phase high pressure liquid chromatography. Overall recovery of IL 2 activity ranged from 70 to 100% of initial activity and yielded 2 X 10(6) or greater units of IL 2 per 15 liters of serum-free MLA144 conditioned medium. The specific activity of purified IL 2 ranged from 0.5 to 1 X 10(8) IL 2 U/mg protein. The purified IL 2 showed four molecular species when analyzed by two-dimensional isoelectric focusing-SDS-polyacrylamide gel electrophoresis. Each of the four molecular forms was active in the bioassay for IL 2 activity. Three molecular forms had apparent m.w. of 16,000 but different isoelectric points of 5.9, 6.3, and 6.7. One molecular form had an apparent m.w. of 15,000 and an isoelectric point of 7.2. The most abundant form of IL 2 had an apparent m.w. of 16,000 and an isoelectric point of 6.3. The purified IL 2 supported the growth of IL 2-dependent lymphocytes to a greater extent than did the same level of crude IL 2-containing MLA144 conditioned medium. The ability to purify large amounts of IL 2 by a rapid and efficient procedure will be of great help in both biochemical and immunologic studies of this lymphokine.

Considerations sur la theorie de la voltammetrie cyclique sur des microelectrodes ayant une epaisseur inferieure a 1 μm, dans le cas de processus aux electrodes ou l'etape du transfert d'electrons controle la cinetique. Exemple dans le cas de l'oxydation a un electron du ferrocene en cation ferrocenium

The primary structure of human erythrocyte carbonic anhydrase C has been determined. The single polypeptide chain contains 259 amino acid residues devoid of disulfide bridges. The experimental approach has involved restriction of the action of trypsin to arginyl bonds by amidination of the lysyl side chains. The six tryptic fragments obtained have been separated and sequenced by manual techniques. During the sequence work on human carbonic anhydrase C, 3 very easily deamidated asparagine residues were noted, all occurring in -Asn-Gly- sequences. The deamidation which takes place even under normal conditions of peptide preparation seems to be associated with a beta-aspartyl shift. A few minor differences existing between our structure and the results from another laboratory are discussed. A brief comparison is made with the primary structures of other carbonic anhydrases with regard to the function of some amino acid residues in the active site of the enzymes.

In 1928, studies on the rate of escape of carbon dioxide from hemolyzed blood led to the recognition of a red cell substance catalyzing the reversible hydration of CO2 [12]. By 1932, the substance was shown to be an enzyme and given the name carbonic anhydrase [26, 27]. In the following 10 years the enzyme was partially purified [28], shown to be a zinc-containing enzyme [15, 16] having a molecular weight of about 30,000 [33] and to contain one zinc ion per enzyme molecule [37]. World War II and its aftermath interrupted research on carbonic anhydrase for almost 20 years, but in the early 60’s researchers in Sweden [23, 30] reported several homogenous preparations of the enzyme which differed in electrophoretic mobility and specific activity. These observations were quickly confirmed by others [18, 34, 35] with the resulting realization that human erythrocytes contained several different enzyme forms catalyzing the reversible hydration of carbon dioxide.