Synthesis of novel immunologically active tripalmitoyl-S-glycerylcysteinyl lipopeptides as useful intermediates for immunogen preparations

The synthesis and characterization of lipopeptides consisting of the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteine (Pam3Cys-OH) and different peptide segments and/or spacer molecules is described. Pam3Cys-peptides, which are derived from the immunologically active N-terminus of bacterial lipoprotein, were obtained either by solution or solid phase peptide synthesis. In particular, the amphiphilic and water-soluble lipohexapeptides Pam3Cys-Ser-(Lys)4 and Pam3Cys-Ser-(Glu)4 proved to be potent macrophage and B-cell activators and non-toxic, non-pyrogenic immune adjuvants in combination with or covalently linked to antigens and haptens.     

Further characterization and chemical purity assessment of the human erythrocyte glucose transporter preparation

Chemical and functional purity of the human erythrocyte glucose transporter preparation obtained by DEAE column chromatography after octyl glucoside solubilization was assessed. The cytochalasin B binding capacity of the preparation indicates that the preparation is 60-85% functional glucose transporter. Gel filtration chromatography on TSK 250 column separates this preparation into at least three major peptide fractions, namely, P0, P1 and P2, with apparent Mr of approx. 80 000, 43 000 and 17 000, respectively. When the preparation is photolabelled with [3H]cytochalasin B prior to the separation only P0 and P1 are labelled. Exposure of the preparation to octyl glucoside or to ultraviolet light irradiation results in an increase in P0 in a time-dependent manner with a concomitant and proportional reduction in P1, without affecting P2 appreciably. For individual preparations, relative abundance of P0 and P1 vary widely in a reciprocal fashion, while that of P2 is practically fixed at approx. 10% of the total protein. The specific activity of cytochalasin B binding of each preparation correlates linearly with the relative abundance of P1 of the preparation, which gives a calculated specific binding activity of 22 nmol/mg protein for this fraction. These results indicate that P1 and P0 are native and denatured transporter, respectively, while P2 is contaminating protein impurities. These results demonstrate that the glucose transporter preparation contains approx. 10% of nontransporter protein impurities, with a varying amount (up to 30%) of denatured transporter, and that the transporter free of the chemical impurities and the denatured transporter can be obtained by a gel filtration chromatography of this preparation.     

Effects of prostaglandins E2 and F2 alpha on progesterone metabolism by rat granulosa cells

Rat granulosa cells were cultured with or without PGE2 and/or PGF2 alpha. Accumulation of endogenous progesterone and 20 alpha-hydroxy-4-pregnen-3-one was determined. Additionally, [4-14C]progesterone metabolism was assessed. PGE2 increased progesterone accumulation, in part, by decreasing progesterone catabolism to 20 alpha-reduced progestins. In contrast, PGF2 alpha stimulated 20 alpha-hydroxysteroid dehydrogenase activity, thus increasing progesterone catabolism. Combined treatment with PGE2 and PGF2 alpha augmented progesterone accumulation to levels above controls but below those attained with PGE2 alone. These data indicate that PGE2 and PGF2 alpha exert opposite effects on progesterone production and catabolism and that the ratio of PGE2 to PGF2 alpha in the local granulosa cell milieu may be of importance in determining overall progesterone output.     

Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities

The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the "monomer" of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

Analyse von zwei Legumin-Genfamilien der Ackerbohne (Vicia faba L.)

DNA clones representing two subfamilies A and B of legum in genes and a recombinant phage containing a complete legumin B gene have been isolated and characterized by DNA sequencing. A DNA fragment harbouring the legumin B gene and adjacent sequences was used for Ti-mediated transfer into tobacco cells.     

G-11 staining in Turner's syndrome with mos 45,X/46,X,r(?)

Mos 45,X/46,X,r(?) in 4 patients with Turner's syndrome and no signs of virilization, and in one pair of monozygotic twins, one of them with clitoral hypertrophy, was studied using combined cytogenetic techniques and specially G-11 staining for the characterization of the X or Y origin of the rings. In all 6 patients the ring was G-11 positive, attesting its Y origin. Both twins were operated and bilateral streak gonads with a bilateral nodule of testicular tissue were found. Similar small rings were also studied in one patient with mos 46,XX/46,X,r(X) and in one nonvirilized Turner's syndrome patient with a larger ring; in these two cases the ring was G-11 negative. It seems that the small rings occasionally found in Turner's syndrome are more frequently from Y origin and therefore prophylactic gonadectomy should be considered.     

Survival of Bacteria and Fungi in Relation to Water Activity and the Solvent Properties of Water in Biopolymer Gels

Survival of bacteria (Rhizobium, Agrobacterium, and Arthrobacter spp.), fungal spores (Penicillium sp.), and yeasts (Saccharomyces sp.) was studied in relation to water activity (a_w) and the presence of nutritive solutes. The cells were entrapped in polysaccharide gels, as is done to immobilize cells or enzymes, and then dehydrated. The number of living cells (10¹⁰ g of dry polymer⁻¹) remained constant for periods of storage of >3 years at 28°C when the inocula were kept at an a_w of <0.069. At a_w values between 0.069 and 0.83 the number of survivors diminished more and more rapidly as the a_w was raised. For a given a_w and organism, there were large differences in survival rate as a function of the nutritive solutes used to culture the microorganisms. Low-molecular-weight compounds (with three or five carbon atoms) had a deleterious effect on survival, whereas compounds of higher molecular weight (C₆ to C₁₂) had a protecting effect. Thus, the a_w alone was not a sufficient explanation for the deterioration of the inocula. Survival seemed to be more directly related to some properties of the water in the biopolymer. New concepts such as the discontinuity of properties of water and the point of mobilization of solutes, already proposed by Duckworth and Kelly (J. Food Technol. 8:105-113, 1973) and Seow (J. Sci. Food Agric., 26:535-536, 1975), have been taken into consideration to explain the interactions of water with the biopolymer and their specific effects on the microorganisms.     

Identification of the polypeptide encoded by the URF-1 gene of Neurospora crassa mtDNA

Two peptides, potentially representing antigenic determinants of a proposed gene product, were synthesized. The peptide sequences were deduced from the nucleotide sequence of the unidentified reading frame (URF)1 of the Neurospora crassa mitochondrial genome. Specific antisera to the synthetic peptides were produced. The antibodies recognized a single polypeptide species with an apparent relative molecular mass of about 30 000. The mitochondrial origin of this polypeptide was verified by in vivo labelling experiments in the presence of cycloheximide, as well as by in vitro translation using isolated mitochondria. The chemical identification of the protein was performed by partial radiosequencing of the N-terminal portion of the immunoprecipitated URF-1 product. The amount of URF-1 polypeptide present in N. crassa mitochondria is in the range of 1-2%. The protein is a constituent of the inner envelope of the organelle and probably part of a more complex membrane unit.