Clinical experiences of using a silver hydroalginate dressing in Austria, Switzerland and Germany.

This retrospective study, which involved patients with a range of colonised or infected wounds from 12 central European wound-care centres, found that this dressing combated infection, reduced pain and promoted healing.

Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography.

We have investigated the use of column chromatography for the purification of ACN53, a recombinant adenovirus type 5 encoding the human p53 tumor suppressor protein. Anion exchange, size exclusion, hydrophobic interaction, and metal chelating resins were tested; each was found to have distinct advantages and disadvantages. Based on these data, a rapid method was devised for the purification of ACN53. The resultant product was characterized and compared to cesium chloride density-gradient purified virus by SDS-PAGE, Western blot analysis, absorbance spectrum, total particle-to-infectious particle ratio, expression of p53 gene product in Saos-2 cells, growth inhibition of Saos-2 cells, and contamination by ATCC-293 host cell proteins. The results show that column chromatography offers an alternative to ultracentrifugation for the purification of recombinant adenoviruses for use in human gene therapy trials and other research applications.

Reduced virus load in rhesus macaques immunized with recombinant gp160 and challenged with simian immunodeficiency virus.

As a safe alternative to inactivated and live-attenuated whole-virus SIV vaccines, we have evaluated the potential of SIVmac239 gp160 expressed by recombinant vaccinia virus (vSIVgp160) and baculovirus (bSIVgp160) to protectively immunize rhesus macaques against intravenous (i.v.) infection with pathogenic SIVmac isolates. Macaques were immunized with live vSIVgp160 and/or bSIVgp160 protein partially purified from insect cells. The challenge viruses, propagated in rhesus peripheral blood mononuclear cells, consisted of the molecular clone SIVmac239 and another genetically similar, uncloned isolate, SIVmac251. Although antibodies that bind gp130 were induced in all animals following immunization with SIVgp160, neutralizing antibodies were undetectable 1 week prior to virus challenge. These results differ from those for macaques vaccinated with inactivated, whole SIV. All animals became infected after i.v. inoculation with 1-10 AID50 of either challenge virus. For animals challenged with SIVmac251, but not those challenged with SIVmac239, the cell-free infectious virus load in plasma of vSIVgp160-primed, bSIVgp160-boosted macaques was significantly lower than in unimmunized controls at 2 weeks postchallenge. Virus virulence, immunization regimen, and challenge with homologous or heterologous virus are factors critical to the outcome of the study. Immunization with surface glycoprotein may not necessarily provide protective immunity against infection but may reduce virus load. The relationship between reduction in virus load by vaccination and delay in onset of disease remains to be determined.

Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development.

The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gel-filtration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd approximately 2 x 10(-9) M).

Characterization of group specific antibodies in primates: studies with SIV envelope in macaques.

Sera from SIV-infected macaques were found to contain antibodies that reacted with conformation-dependent, group-specific determinants on the SIV envelope protein gp130. These conformation-dependent antibodies exhibited virus neutralizing activity; their presence was associated with protection in vaccine studies. The properties of these antibodies are quite similar to those that have been identified in sera from HIV-infected human subjects. These data suggest that the SIV envelope gp130 remains a candidate for subunit vaccine studies.

Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons.

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gp120. The objective of the studies presented here was to test whether a native recombinant gp120 (rgp120) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gp120 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgp120SF2 but not with denatured, nonglycosylated env 2-3SF2. After repeated exposure to rgp120SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgp120 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.

Functional and immunological characterization of SIV envelope glycoprotein produced in genetically engineered mammalian cells.

Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immune responses in infected hosts. Macaque simian immunodeficiency virus (SIVmac) is a T-lymphocytopathic lentivirus which causes an AIDS-like disease in rhesus macaques. The envelope gene of SIVmac encodes a precursor glycoprotein (gp160) which is cleaved into an external domain (gp130) and a transmembrane domain (gp32). To investigate the functional and immunological properties of the SIV external envelope glycoprotein, we have used genetically engineered mammalian cells to produce recombinant gp130 (rgp130). The rgp130 has the appropriate molecular weight, is glycosylated, and has native conformation as determined by binding to the cell receptor for SIV, the CD4 antigen. Rhesus macaques immunized with purified rgp130 formulated in muramyl dipeptide adjuvant generated high titers of antienvelope antibodies. Antibodies from these macaques were tested for in vitro virus neutralization; very low or undetectable levels of neutralization were observed. In contrast, neutralizing antibodies were readily detected in sera from goats immunized with rgp130. With respect to cell-mediated immunity, proliferative responses to rgp130 were demonstrated in peripheral blood monocyte cells (PBMC) from macaques immunized with the recombinant glycoprotein as well as in PBMC from SIV-infected animals. These results show that rgp130 is functional and immunogenic; the potential of rgp130 for protective immunization remains to be determined.

Neutralization of divergent HIV-1 isolates by conformation-dependent human antibodies to Gp120.

The spectrum of human immunodeficiency virus type 1 (HIV-1) isolates neutralized by antibodies from HIV-1-infected humans is broader than the spectrum of isolates neutralized by sera from animals immunized with purified gp120 subunits. This broader neutralization was due, in part, to the presence of antibodies to conserved gp120 conformational epitopes. Purified conformation-dependent gp120-specific human antibodies neutralized a wider range of virus isolates than human antibodies directed to linear determinants in gp120 and were also responsible for the majority of the gp120-specific CD4-blocking activity of HIV-1-infected human sera. A gp120 subunit vaccine that effectively presents these conformation-dependent neutralization epitopes should protect against a broader range of HIV-1 variants than a vaccine that presents exclusively linear determinants.

Respiration capacity of mitochondria isolated from unfertilized and fertilized sea urchin eggs.

Mitochondria were isolated from unfertilized and fertilized eggs of the sea urchin, Strongylocentrotus purpuratus. Both preparations exhibited coupled adenosine 5'-diphosphate (ADP)-dependent) oxidation of flavin and pyridine-linked substrates and both yielded the expected P:O ratios with these substrates. Highest respiratory control indices (greater than 4.0) were observed when succinate or pyruvate + malate were used as substrates. Mitochondria from unfertilized and fertilized eggs exhibited sensitivity to respiratory and phosphorylation inhibitors and uncouplers and both preparations exhibited cross-over points at sites I, II and III of the respiratory chain. Low-temperature difference spectra revealed a normal complement of cytochromes c, b and aa3, although cytochrome c from unfertilized eggs appears to be more subject to extraction during the course of mitochondrial isolation than does cytochrome c from fertilized eggs. An unidentified pigment absorbing at approx. 570 nm was visible in low-temperature spectra of unfertilized eggs and unfertilized egg mitochondria.

Crystallographic characterization of recombinant human CuZn superoxide dismutase.

Recombinant human CuZn superoxide dismutase as expressed in yeast has been crystallized in three different crystal forms. Hexagonal plates grow from 2.4 M ammonium sulfate, pH 7.5, and belong to the space group P6(3)22, with cell dimensions a = b = 113.5(3), c = 151.5(5) A, and Vm = 2.21 A3/dalton for two dimers per asymmetric unit. At 2.0 M ammonium sulfate, pH 7.5, chunky wedges grow in space group C222(1), a = 205.2(6), b = 166.5(4), c = 145.4(4) A with a Vm of 2.43 A3/dalton for eight dimers per asymmetric unit. With polyethylene glycol 8000, pH 7.5-8.0, hexagonal prisms are obtained with cell dimensions a = b = 197.4(6), c = 43.1(2) A, space group P6, and Vm = 2.53 A3/dalton for three dimers per asymmetric unit. All of these forms diffract to high resolution, are stable to x-rays, and appear suitable for determination of the atomic structure. Crystals of the doubly mutated enzyme (Cys6----Ala, Cys111----Ser) grown from both micro- and macroseeds of the wild type protein demonstrate the feasibility of isomorphous crystallization of site-directed mutants of the cloned parent enzyme for comparative structure-function studies.

Growth restoration of insulin-deficient diabetic rats by recombinant human insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) and insulin stem from a common precursor, are structural homologues, act through similar receptors and elicit insulin-like and growth-promoting effects in vitro and in vivo. Serum IGF-I levels are controlled by growth hormone, insulin and nutrition. Insulin-deficient growth-arrested diabetic animals have reduced serum IGF-I levels which are restored towards normal by insulin but not by growth-hormone treatment. Here we show that normal growth of diabetic rate is restored by infusion of recombinant human (rh)IGF-I without normalization of the blood sugar level and that insulin acts via an increase of IGF-I synthesis on growth of diabetic rats. We describe a new mechanism of endocrine control of growth in which IGF-I is the major stimulator at the cellular level. Growth hormone and insulin act mainly by modulating the hepatic synthesis of IGF-I.

Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library.

The molecular cloning and nucleotide sequence of the cDNA for human Cu/Zn superoxide dismutase (SOD) is reported. The tacI promoter has been used to direct the synthesis in E. coli of this SOD which is soluble, stable, and of normal specific activity. The N-terminal methionine is removed from this protein. A construction with a ribosome binding site identical to that of the lacz gene 5' of the initiator methionine codon, resulted in low levels of SOD. An in vitro mutagenesis procedure was used to randomize the four nucleotides preceding the initiator methionine codon and the silent third positions of the codons specifying the second and third amino acids. Analysis of a sample of 500 clones showed that ca. 25 clones synthesised 5% or more of soluble cell protein as SOD. The nucleotide sequences of high level expressors showed a predominance of A and T residues in the variable positions 5' of the initiator methionine codon. An SOD mutant (ala4----gln) was discovered during the sequencing and shown to lack dismutation activity. Secondary structure predictions for the 5' regions of the mRNAs from high and low level expressors support the hypothesis that initiation of translation is much reduced if part of the region complementary to 16s rRNA is base paired in a stem structure.

Arachidonic acid and other fatty acids inhibit secretion from sea urchin eggs.

Massive secretion at the egg surface follows fertilization of sea urchin eggs or parthenogenetic activation by the calcium ionophore A23187. The secretory products are used to construct the fertilization envelope around the egg. Arachidonic acid prevents the raising of the fertilization envelope induced by either sperm or A23187. We developed a secretion assay based on the ability of A23187 to raise fertilization envelopes from the surface of unfertilized eggs. Arachidonate delays the onset of this reaction in a dose-dependent fashion. 5 microM arachidonate produces a two-fold delay in the standard assay. In contrast, the propagation of secretion over the surface of the egg is unaffected at all concentrations that have been tested. Some closely related fatty acids (e.g. 11, 14, 17 C20:3 and linoleate, 9, 12 C18:2) share with arachidonate the ability to inhibit secretion, whereas others (e.g., 8, 11, 14 C20:3 and linolenate, 9, 12, 15 C18:3) do not. The results are not easily reconciled with a cyclooxygenase- or a lipoxygenase-mediated action. Despite the sensitivity of this phenomenon to small changes in fatty acid structure, it is suggested that the fatty acids exert their effect by altering the structure or dynamics of the membrane lipid bilayer.

Calcium-induced decrease in membrane fluidity of sea urchin egg cortex after fertilization.

Fertilization of the sea urchin egg is a dramatic example of cell activation resulting from the interaction of an external stimulus, the spermatozoon, with the cell surface. Growing and quiescent cells may have different membrane states. Here we report membrne fluidity measurements on a surface membrane fraction, the cortex, isolated from unfertilized and fertilized eggs. The fluidity of the fertilized egg cortex, measured by electron spin resonance (ESR) spectroscopy using 5-doxylstearate as a probe, is less than that of the unfertilized cortex. In the intact egg the intracellular CA2+ to the cortex fraction isolated from unfertilized eggs triggers a fluidity decrease in vitro. The fluidity decrease seems to represent a Ca2+-induced change in membrane structure rather than a direct interaction of Ca2+ with phospholipid headgroups.

Bulk membrane fluidity increases after fertilization or partial activation of sea urchin eggs.

Membrane fluidity increases within 10 min after fertilization of Strongylocentrotus purpuratus and Lytechinus pictus eggs as detected by a decrease in order parameter of the spin label fatty acid 5-doxylstearate. The magnitude of this change is proportional to the fraction of fertilized eggs present. The order parameter decreases when unfertilized eggs are partially activated by ammonia treatment but does not change when the extracellular coats are removed. The increase in membrane fluidity, therefore, appears to be a "late" event in the fertilization program. The increase in fluidity is confined to the polar head group region of the membrane bilayer. Spectral and autoradiographic analyses indicate that the spin label resides in lipid bilayers throughout the cell. Thus, these measurements apply to bulk cell membranes. Detectable changes in lipid composition do not occur shortly after fertilization.

Cholesterol levels and plasma membrane fluidity in 3T3 and SV101-3T3 cells.

Polyene antibiotics such as filipin selectively inhibit wheat germ agglutinin-induced agglutination of transformed and malignant cells compared to normal cells (Hatten ME, Burger MM: Biochemistry 18: 739, 1979). Since filipin binds specifically to cholesterol, we measured cholesterol levels in 3T3 cells and SV101-3T3 cells. SV101-3T3 cells contained 50-100% more cholesterol per cell than 3T3 cells. Both cell types were starved for cholesterol by growth in lipid-depleted medium plus 25-hydroxycholesterol. The cholesterol level of SV101-3T3 cells decreased by 30-50%, while the level in 3T3 cells remained constant. Filipin-stained SV101-3T3 cells revealed bright patches of filipin under fluorescence microscopy. These patches were absent in 3T3 cells and in SV101-3T3 and 3T3 cells starved for cholesterol. We selectively labeled plasma membranes of these cells with a spin label analog of phosphatidylcholine. The spin label indicated differences in plasma membrane fluidity that may be related to the different cholesterol levels in 3T3 and SV101-3T3 cells.

Similarities in the membrane fluidity of 3T3 and SV101-3T3 cells and its relation to concanavalin A- and wheat germ agglutinin-induced agglutination.

Intact, viable ultransformed 3T3 and transformed SV101-3T3 cells were labeled with fatty acid spin labels and with 2,2,6,6-tetramethylpiperidine-1-oxyl in order to measure the fluidity properties of membrane lipids. Both cell types were grown in regular calf serum and in a lipid-depleted serum supplemented with either oleate or elaidate. The temperature dependence of the spectra obtained revealed inflections that correlate with the temperature below which agglutination with concanavalin A is inhibited, and another inflection that correlates with the temperature below which agglutination with wheat germ agglutinin is inhibited, suggesting that (a) the lipid phase(s) in the vicinity of the receptor(s) for these two lectins differ, and (b) a fluid membrane in the vicinity of the lectin receptor(s) is necessary for agglutination with either concanavalin A or wheat germ agglutinin. Studies with a partially characterized plasma membrane fraction suggest that the plasma membrane fluidity parameters closely resemble those of the intact cell. 3T3 and SV101-3T3 cells show virtually identical fluidity profiles by all of the tests we have applied.

Fertilization-induced changes in membrane fluidity of sea urchin eggs.

By use of a spin label fatty acid, 5-doxylstearate, an increase in bulk membrane fluidity was observed after fertilization of two species of sea urchin eggs. Eggs partially activated by ammonia showed a similar effect. The data suggest that a structural change involving membrane lipids accompanies activation.

Spin labeling of a cysteine residue of the Escherichia coli outer membrane lipoprotein in its membrane environment.

A method was developed to attach a spin label to a specific site on the structural lipoprotein of the Escherichia coli outer membrane in situ. This method takes advantage of the fact that the outer membrane of wild-type E. coli contains few residues reactive towards sulfhydryl reagents. A mutant E. coli strain has been isolated [Suzuki, H., Nishimura, Y., Iketani, H., Campisi, J., Hirashima, A., Inouye, M. & Hirota, Y. (1976) J. Bacteriol. 127, 1494-1501] in which the second position from the carboxy terminus of the lipoprotein is changed from arginine into a cysteine residue. The membrane fraction of this mutant was treated with N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)maleimide in the presence of EDTA and 2-mercaptoethanol. Spin label was found to be preferentially incorporated into the lipoprotein. The spectrum of the spin-labeled membrane shows two components, both arising from spin label at the same site near the carboxy terminus. The strongly immobilized component has a maximum hyperfine splitting value of 53 G, and the weakly immobilized component, 37 G. A fraction of the lipoprotein is covalently bound to the peptidoglycan layer through its carboxy-terminal lysine; the spectrum of the isolated bound form of the lipoprotein was identical to that of the free form. When the matrix protein, the other major outer membrane protein, was removed by mutation, the spectrum of the lipoprotein was altered, suggesting that these two proteins are closely associated.